[Pharyngeal acid load and different functional endoscopy findings]. / Pharyngeale Säurebelastung bei unterschiedlichen Befunden der funktionellen Endoskopie.

The findings of functional endoscopy (upper esophageal sphincter insufficiency, cardia insufficiency, esophagitis, gastric heterotopia, axial sliding hernia, and visible aerosols) can be traced back to pharyngeal acid exposure by oropharyngeal pH measurement. Significantly increased pharyngeal acid loads are seen in gastric heterotopy and axial sliding hernia. For all measured statistics, the pharyngeal acid load is in the pathological or even very pathological range. The value of functional endoscopy in the context of laryngopharyngeal reflux diagnosis is clearly documented. The findings "heterotopic gastric mucosa" and "axial sliding hernia" may cause marked airway symptoms and a pathogenetic relationship with otorhinolaryngologic reflux-associated symptoms must be postulated for these entities.

[Relationship between functional endoscopy and impedance-pH measurement. German version]. / Beziehung zwischen Funktionsendoskopie und Impedanz-pH-Metrie.

Classic gastroenterological diagnostic tools are proving increasingly insufficient for analyzing the complex causal relationship between extra-esophageal symptoms and presumed pathological reflux activity. Some new methodological approaches, such as functional endoscopy (videopanendoscopy, VPE), are considered to be helpful. However, there are currently no data objectively verifying the usefulness of this method. In a pilot study, a good correlation between the reflux symptom index (RSI) and endoscopic findings was shown. Impedance-pH measurement is considered to be the "gold standard" in esophageal reflux disease diagnostics. Therefore, the relationship between endoscopic findings and the results of impedance-pH monitoring are now studied in patients with extra-esophageal reflux symptoms. The investigation demonstrates that the pathological findings of VPE correlate well with impedance-pH measurements in terms of the parameters "number of reflux episodes", "fraction time", and "DeMeester score".

Relationship between functional endoscopy and impedance-pH measurement.

Classic gastroenterological diagnostic tools have proven to be insufficient in identifying the causal relationship between extra-esophageal symptoms and presumed pathological reflux activity. Some new methodological approaches, such as functional endoscopy (video panendoscopy, VPE), are considered to be helpful. However, there are currently no data objectively verifying the success of this method. In a previous study, we found a good correlation between the reflux symptom index (RSI) according to Belafsky and endoscopic findings. Impedance-pH measurement is considered to be the gold standard in esophageal reflux disease diagnostics. Therefore, the relationship between endoscopic findings and the results of impedance-pH monitoring is now studied in patients with extra-esophageal reflux symptoms. The pathological findings of the VPE correlate with impedance-pH measurements regarding the parameters "number of reflux episodes," "fraction time," and "DeMeester score."

[Importance of functional endoscopy for diagnostics of extraesophageal reflux : relationship of Belafsky's reflux symptom index and functional endoscopic data]. / Stellenwert der Funktionsendoskopie für die Diagnostik des extraösophagealen Refluxes : Beziehung des Reflux-Symptom-Index zu funktionsendoskopischen Daten.

BACKGROUND: Extraesophageal reflux exists as shown by scientific data. The underlying pathophysiology is not yet exactly known. Functional endoscopy seems to be a promising new diagnostic instrument. This study determined the relationship between functional endoscopy and Belafsky's reflux symptom index (RSI). METHODS: In this study 71 patients were prospectively included and underwent a functional endoscopic examination followed by 6 months of proton pump inhibitor (PPI) therapy. Symptoms were scored using Belafsky's RSI after endoscopic examination (before treatment) and after 3 and 6 months of PPI therapy. RESULTS: After 3 and 6 months of PPI-therapy significant decreases in the RSI were found. The functional endoscopy characteristics which were expected to remain constant were controlled after 6 months and found to be nearly 100 % reproducible. Patients profit from PPI- therapy when reflux is detected by endoscopy even when RSI was initally normal. Functional endoscopy seems to be a useful instrument to detect extraesophageal reflux.

White matter injury and autistic-like behavior predominantly affecting male rat offspring exposed to group B streptococcal maternal inflammation.

The impact of the group B streptococcus (GBS)-induced maternal inflammation on offspring's brain has not yet been investigated despite GBS being one of the most frequent bacteria colonizing or infecting pregnant women. According to our hypothesis GBS-induced maternal immune activation plays a role in offspring perinatal brain damage and subsequent neurodisabilities such as autism. Using a new preclinical rat model of maternal inflammation triggered by inactivated GBS, we demonstrated placental, neuropathological and behavioral impacts on offspring. GBS-exposed placentas presented cystic lesions and polymorphonuclear infiltration located within the decidual/maternal side of the placenta, contrasting with macrophagic infiltration and necrotic areas located in the labyrinth/fetal compartment of the placenta after lipopolysaccharide-induced maternal inflammation. Brain damage featured lateral ventricles widening, predominately in the male, reduction of periventricular external capsules thickness, oligodendrocyte loss, and disorganization of frontoparietal subcortical tissue with no glial proliferation. Autistic hallmarks were found in offspring exposed to GBS, namely deficits in motor behavior, social and communicative impairments, i.e. profound defects in the integration and response to both acoustic and chemical signals that are predominant modes of communication in rats. Surprisingly, only male offspring were affected by these combined autistic-like traits. Our results show for the first time that materno-fetal inflammatory response to GBS plays a role in the induction of placental and cerebral insults, remarkably recapitulating cardinal features of human autism such as gender dichotomy and neurobehavioral traits. Unlike other models of prenatal inflammatory brain damage (induced by viral/toll-like receptor 3 (TLR3) or Gram-negative/TLR4), maternal inflammation resulting from GBS/TLR2 interactions induced a distinctive pattern of chorioamnionitis and cerebral injuries. These results also provide important evidence that beyond genetic influences, modifiable environmental factors play a role in both the occurrence of autism and its gender imbalance.

Histone deacetylases: novel targets for prevention of colitis-associated cancer in mice.

OBJECTIVE: Inhibition of histone deacetylases, well known for its antiproliferative efficacy in vivo, was recently shown to ameliorate inflammation in experimental colitis. Since inflammatory bowel disease is associated with an increased risk of developing colon cancer, here the combined anti-inflammatory and proapoptotic efficacy of histone deacetylase inhibitors was studied in mouse models. METHODS: The novel histone deacetylase inhibitor ITF2357 was compared with suberoylanilide hydroxamic acid in models of experimental colitis. Effects on tumour growth were studied after treatment of mice with azoxymethane and dextran sulphate sodium, and in interleukin 10 (IL10) knockout mice, respectively. Possible underlying mechanisms involving apoptosis and nuclear factor (NF)-kappaB activation were addressed by flow cytometry and western blot. RESULTS: In dextran sulphate sodium- and trinitrobenzene sulphonic acid-induced colitis, treatment with ITF2357 was superior to suberoylanilide hydroxamic acid as shown by macroscopic and histological amelioration of inflammation, by reduced production of interferon gamma (IFN gamma) and by increased production of IL10. In both models of inflammation-mediated tumourigenesis, inhibition of histone deacetylases resulted in a significant suppression of tumour growth in terms of size and number, along with reduced signs of inflammation. As for potential mechanisms of ITF2357 action, increased acetylation of histone 3, reduced production of IFN gamma and enhanced apoptosis in lamina propria mononuclear cells were found to accompany a histone deacetylase-dependent activation of NF-kappaB. CONCLUSIONS: These results indicate that inhibition of histone deactylases can attenuate inflammation-mediated tumour growth, which is paralled by an inhibition of NF-kappaB. Thus histone deacetylase inhibitors provide a promising strategy that combines anti-inflammatory and proapoptotic modes of action.

Embryonic and postnatal development of the serotonergic raphe system and its target regions in 5-HT1A receptor deletion or overexpressing mouse mutants.

The neurotransmitter 5-HT regulates early developmental processes in the CNS. In the present study we followed the embryonic and postnatal development of serotonergic raphe neurons and catecholaminergic target systems in the brain of 5-HT1A receptor knockout (KO) and overexpressing (OE) in comparison with wild-type (WT) mice from embryonic day (E) 12.5 to postnatal day (P) 15.5. Up to P15.5 no differences were apparent in the differentiation and distribution of serotonergic neurons in the raphe area as revealed by the equal number of serotonergic neurons in the dorsal raphe in all three genotypes. However, the establishment of serotonergic projections to the mesencephalic tegmentum and hypothalamus was delayed at E12.5 in KO and OE animals and projections to the cerebral cortex between E16.5 and E18.5 were delayed in OE mice. This delay was only transient and did not occur in other brain areas including septum, hippocampus and striatum. Moreover, OE mice caught up with WT and KO animals postnatally such that at P1.5 serotonergic innervation of the cortex was more extensive in the OE than in KO and WT mice. Tissue levels of 5-HT and of its main metabolite 5-hydroxyindoleacetic acid as well as 5-HT turnover were considerably higher in brains of OE mice and slightly elevated in KO mice in comparison with the WT, starting at E16.5 through P15.5. The initial differentiation of dopaminergic neurons and fibers in the substantia nigra at E12.5 was transiently delayed in KO and OE mice as compared with WT mice, but no abnormalities in noradrenergic development were apparent in later stages. The present data indicate that 5-HT1A receptor deficiency or overexpression is associated with increased 5-HT synthesis and turnover in the early postnatal period. However, they also show that effects of 5-HT1A KO or OE on the structural development of the serotonergic system are at best subtle and transient. They may nonetheless contribute to the establishment of increased or reduced anxiety-like behavior, respectively, in adult mice.

Morphine priming in rats with chronic inflammation reveals a dichotomy between antihyperalgesic and antinociceptive properties of deltorphin.

We previously showed that prolonged morphine treatment and chronic inflammation both enhanced delta opioid receptor (deltaOR) cell surface density in lumbar spinal cord neurons. Here, we sought to determine whether administration of morphine to rats with chronic inflammation would further increase the bio-availability of deltaOR, and thereby the analgesic properties of the deltaOR agonist deltorphin, over that produced by inflammation alone. We found that chronic inflammation produced by injection of complete Freund's adjuvant (CFA) into the hind paw resulted in a bilateral increase in the binding and internalization of fluorescent deltorphin in neurons of the lumbar spinal cord as did prolonged morphine treatment [Morinville A, Cahill CM, Aibak H, Rymar VV, Pradhan A, Hoffert C, Mennicken F, Stroh T, Sadikot AF, O'Donnell D, Clarke PB, Collier B, Henry JL, Vincent JP, Beaudet A (2004a) Morphine-induced changes in delta opioid receptor trafficking are linked to somatosensory processing in the rat spinal cord. J Neurosci 24:5549-5559]. This effect was accompanied by an increase in the antinociceptive efficacy of intrathecal deltorphin as measured using the tail-flick test. Treatment of CFA-injected rats with morphine decreased the cell surface availability of deltaOR in neurons of the dorsal horn of the lumbar spinal cord as compared with treatment with CFA alone. Behaviorally, it significantly enhanced the antihyperalgesic effects of deltorphin (plantar test; % maximum possible antihyperalgesic effect (MPAHE)=113.5%+/-32.4% versus 26.1%+/-11.6% in rats injected with CFA alone) but strongly reduced the antinociceptive efficacy of the drug (tail-flick test; % maximum possible antinociceptive effect (MPE)=29.6%+/-3.6% versus 66.6%+/-6.3% in rats injected with CFA alone) suggesting that the latter, but not the former, is linked to the deltaOR trafficking events observed neuroanatomically. These results demonstrate that in chronic inflammation, the antihyperalgesic effects of deltaOR agonists may be enhanced by morphine pre-treatment. They also reveal a dichotomy between mechanisms underlying antihyperalgesic and antinociceptive effects of deltaOR agonists.

Toll-like receptor expression and response to specific stimulation in adipocytes and preadipocytes: on the role of fat in inflammation.

Data in this study indicate that both adipocytes and preadipocytes express abroad set of TLRs and they also respond to specific stimulation by cytokine production. The may be of relevance to Crohn's disease and a suggests a closer link between adipose tissue and innate immunity.

Activation of annexin A1 signalling in renal fibroblasts exerts antifibrotic effects.

AIM: The anti-inflammatory protein annexin A1 (AnxA1) and its formyl peptide receptor 2 (FPR2) have protective effects in organ fibrosis. Their role in chronic kidney disease (CKD) has not yet been elucidated. Our aim was to characterize the AnxA1/FPR2 system in models of renal fibrosis. METHODS: Rats were treated with angiotensin receptor antagonist during the nephrogenic period (ARAnp) to induce late-onset hypertensive nephropathy and fibrosis. Localization and regulation of AnxA1 and FPR2 were studied by quantitative real-time PCR and double labelling immunofluorescence. Biological effects of AnxA1 were studied in cultured renal fibroblasts from AnxA1(-/-) and wild-type mice. RESULTS: Angiotensin receptor antagonist during the nephrogenic period kidneys displayed matrix foci containing CD73(+) fibroblasts, alpha-smooth muscle actin (a-SMA)(+) myofibroblasts and CD68(+) macrophages. TGF-ß and AnxA1 mRNAs were ~threefold higher than in controls. AnxA1 was localized to macrophages and fibroblasts; myofibroblasts were negative. FPR2 was localized to fibroblasts, myofibroblasts, macrophages and endothelial cells. AnxA1 and FPR2 immunoreactive signals were increased in the foci, with fibroblasts and macrophages expressing both proteins. AnxA1(-/-) fibroblasts revealed higher α-SMA (sevenfold) and collagen 1A1 (Col1A1; 144-fold) mRNA levels than controls. Treatment of murine WT fibroblasts with TGF-ß (22.5 ng mL 24 h(-1)) increased mRNA levels of α-SMA (9.3-fold) and Col1A1 (fourfold). These increases were greatly attenuated upon overexpression of AnxA1 (1.5- and 1.7-fold, respectively; P < 0.05). Human fibroblasts reacted similarly when receiving the FPR2 inhibitor WRW4. CONCLUSION: Our results demonstrate that AnxA1 and FPR2 are abundantly expressed in the renal interstitium and modulate fibroblast phenotype and extracellular matrix synthesis activity.

Long-term effects of fundoplication in children with chronic airway diseases.

BACKGROUND: Association between chronic airway diseases (CAD) and gastroesophageal reflux disease (GERD) is well described, but causality has not yet been conclusively established. This study evaluates the therapeutic significance of laparoscopic Thal fundoplication in children with CAD and diagnosed GERD. METHODS: We performed a retrospective analysis of 182 neurologically nonimpaired children, all with medically refractory CAD and GERD undergoing laparoscopic Thal fundoplication. The clinical response, ability to wean oral and inhaled medication and satisfaction with postoperative results were evaluated. RESULTS: Main symptoms disappeared completely in 68.7% of patients and were markedly improved in a further 22% of patients following surgery. Complete discontinuation of medication was achieved in 70.1-96.4% of cases and reduced in a further 1.8-23.5%. One intraoperative complication occurred (gastric perforation), however no conversion to laparotomy was necessary. Postoperative Dumping Syndrome occurred in 1% of cases and was managed dietetically. Prolonged postoperative dysphagia occurred in 4.3% of patients, but disappeared within 8 weeks in all but one case. CONCLUSIONS: Our study suggests that Thal fundoplication in neurologically nonimpaired children with CAD and documented GERD is effective and safe. Children unresponsive to preoperative medical management showed significant improvement in airway symptoms together with a marked reduction in the need for medication. We conclude that laparoscopic Thal fundoplication represents a significant treatment worthy of consideration in this group of patients.

Intracellular dynamics of sst5 receptors in transfected COS-7 cells: maintenance of cell surface receptors during ligand-induced endocytosis.

Internalization of G protein-coupled receptors is crucial for resensitization of phosphorylation-desensitized receptors, but also for their long term desensitization through sequestration. To elucidate the mechanisms regulating cell surface availability of the somatostatin (SRIF) receptor subtype sst5, we characterized its internalization properties in transfected COS-7 cells using biochemical, confocal microscopic, and electron microscopic techniques. Our results demonstrated rapid and efficient sequestration of specifically bound [125I]Tyr0-D-Trp8-SRIF (up to 45% of bound radioactivity). Combined immunocytochemical detection of sst5 and visualization of a fluorescent SRIF analog by confocal microscopy revealed that whereas the internalized ligand progressively clustered toward the cell center with time, immunoreactive receptors remained predominantly associated with the plasma membrane. The preservation of cell surface receptors was confirmed by binding experiments on whole cells revealing a lack of saturability of [125I]Tyr0-D-Trp8-SRIF binding at 37 C. Binding was rendered saturable by the drug monensin, showing that receptor recycling played a key role in the preservation of cell surface receptors. Electron microscopy demonstrated that in addition to receptor recycling, internalization of receptor-ligand complexes triggered a massive recruitment of sst5 receptor molecules from intracellular stores to the membrane. This combination of recycling and recruitment of spare receptors may protect sst5 from long term down-regulation through sequestration and, therefore, facilitate extended SRIF signaling.

Immunohistochemical distribution of the somatostatin receptor subtype 5 in the adult rat brain: predominant expression in the basal forebrain.

Somatostatin exerts its actions by means of a family of G protein-coupled receptors, five of which have so far been cloned. Whereas mRNAs for receptor subtypes sst(1)-sst(4) have been unequivocally localized in the brain, the data concerning the fifth subtype, sst(5), are contradictory. Moreover, whereas sst(1) and sst(2A) receptor proteins have been localized by immunohistochemistry, the distribution of sst(3)-sst(5) receptor proteins and/or subtype-specific binding remains to be determined in the central nervous system. In the present study, we investigated the distribution of immunoreactive sst(5) in adult rat brain and pituitary and demonstrated the presence of this receptor protein in the central nervous system by using an affinity-purified antibody generated against the C-terminus of the receptor. The specificity of the antibody for sst(5) was established by immunoblotting experiments on membranes prepared from cells transfected with cDNA encoding different somatotropin release inhibiting (SRIF) receptor subtypes as well as from anterior pituitary. In both systems, the antibody specifically recognized a band at approximately 50 kDa molecular mass, corresponding well to the reported size of the cloned receptor (48 kDa). Immunofluorescence in COS-7 cells transfected with individual SRIF receptor subtypes as well as in sections of rat pituitary demonstrated the antibody's applicability to the immunohistochemical detection of sst(5) receptors. In rat brain sections, sst(5) immunoreactivity was predominantly associated with neuronal perikarya and primary dendrites. Immunolabeling was most prominent in the olfactory tubercle, islands of Calleja, diagonal band of Broca, substantia innominata, and magnocellular preoptic nucleus of the basal forebrain as well as in the reticular nucleus of the thalamus. Other, less intensely labeled areas included the cerebral cortex, hippocampus, amygdala, preoptic area as well as the lateroanterior nucleus of the hypothalamus. The present findings provide the first characterization of the anatomic distribution of sst(5) receptors in the rat brain. They demonstrate a prominent expression of these receptors in the basal forebrain, suggesting that they may be involved in the mediation of somatostatin effects on the sleep-wake cycle through their association with cortically projecting subcortical systems.

Somatostatin in the prepacemaker nucleus of weakly electric fish, Apteronotus leptorhynchus: evidence for a nonsynaptic function.

Neuropeptides are widely distributed throughout the nervous system and exert a large number of heterogeneous functions. While they are synthesized in the soma, release is thought to take place in axonal terminals of neurons. A good model system to investigate the role of peptides in the nervous system is provided by the central posterior/prepacemaker nucleus (CP/PPn) of pacemaker nucleus (Pn), a medullary cell group controlling the electric organ discharge (EOD). Previous immunocytochemical and in situ-hybridization studies employing topographical criteria indicated that PPn neurons may express the neuropeptide somatostatin (SS). In the present study, we unambiguously identified PPn neurons by in vitro tract tracing. By combining this technique with SS immunocytochemistry, we found that a large portion of retrogradely labelled PPn neurons exhibited SS-like immunoreactivity (72-89%, n = 708 cells in 10 fish examined). Surprisingly, however, neither the proximal PPn axons nor anterogradely labelled terminals innervating the Pn displayed significant amounts of SS-like immunolabelling (n = 10 fish examined in each experiment). These results and the lack of SS binding sites in the Pn [82] suggest that SS expressed by PPn cells is not synaptically released at the target site of their axons, the Pn, but acts via a nonsynaptic mechanism in the CP/PPn proper.

The postembryonic development of somatostatin immunoreactivity in the central posterior/prepacemaker nucleus of weakly electric fish, Apteronotus leptorhynchus: a double-labelling study.

The neuropeptide somatostatin (SS) is widely distributed in both the central and peripheral nervous system of vertebrates. Its widespread distribution is paralleled by a large variety of diverse functions. While embryonic and perinatal development of SS-like immunoreactivity have been well examined, little is known about the postnatal development of this neuropeptide. Since, in teleosts, neurogenesis persists in many brain regions during adulthood, these vertebrates are well suited to investigate this phenomenon. In the present study, we have, therefore, examined the development of somatostatinergic cells born during adulthood in the central posterior/prepacemaker nucleus (CP/PPn) of Apteronotus leptorhynchus, a weakly electric gymnotiform fish. This was achieved by labelling proliferating cells with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and by simultaneous immunocytochemical detection of SS-like immunoreactivity. SS-like immunoreactivity is adopted in a period between 2 days and 3.5 days after birth. While the number of BrdU-labelled cells in the CP/PPn decreases 10 days after birth, the percentage of double-labelled cells among the BrdU-labelled cells remains with 1.0-7.6% in the period between 3.5 days and 100 days after birth rather constant. This percentage matches well the fraction of SS-positive cells in the total population of cells present in the CP/PPn.

Identification and localization of somatostatin-like immunoreactivity in the cerebellum of gymnotiform fish, Apteronotus leptorhynchus.

Previous studies have shown major mismatches between the localization of the neuropeptide somatostatin of its mRNA and the distribution of somatostatin receptors in the cerebellum of gymnotiform fish. By employing highly sensitive immunohistochemical techniques, we re-examined these mismatches in the gymnotiform fish Apteronotus leptorhynchus. We found somatostatin-like immunoreactivity in the corpus cerebelli, valvula cerebelli, eminentia granularis anterior, eminentia granularis posterior, and transitional zone. While labelled somata were predominantly restricted to a region near the boundary between the granule cell layers and molecular layers, immunopositive processes were distributed throughout large areas within the respective molecular layer. These immunopositive cells appear to represent a novel cell type in the cerebellum of gymnotiform fish.

Expression of somatostatin in neurons of the central posterior/prepacemaker nucleus projecting to the preglomerular nucleus: immunohistochemical evidence for a non-synaptic function.

In the diencephalon of the weakly electric gymnotiform fish Apteronous leptorhynchus, part of the central posterior/prepacemaker nucleus innervates the preglomerular nucleus. A minor population of these neurons expresses immunoreactivity against somatostatin, as has been shown by combining peptide immunohistochemistry with an in vitro tract-tracing technique. In contrast to the expectation, however, this neuropeptide does not appear to be transported along the axons to the projection site, as somatostatin-like immunoreactivity could not be detected in the preglomerular nucleus. It is, therefore, likely that somatostatin expressed in these neurons exerts a non-synaptic function in the region of the central posterior/prepacemaker nucleus itself.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy.

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

Immunohistochemical evidence for synaptic release of glutamate from orexin terminals in the locus coeruleus.

Orexin (Orx or hypocretin) is critically important for maintaining wakefulness, since in its absence, narcolepsy with cataplexy occurs. In this role, Orx-containing neurons can exert their influence upon multiple targets through the brain by release of Orx but possibly also by release of other neurotransmitters. Indeed, evidence was previously presented to suggest that Orx terminals could utilize glutamate (Glu) in addition to Orx as a neurotransmitter. Using fluorescence and confocal laser scanning microscopy, we investigated whether Orx varicosities contain the presynaptic markers for synaptic release of Glu or GABA and come into contact with postsynaptic markers for excitatory synapses within the locus coeruleus of the rat brain. We found that a proportion of the Orx+ varicosities were immunostained for the vesicular transporter for Glu, VGluT2. None were immunostained for vesicular glutamate transporter 1 (VGluT1) or VGluT3 or for the vesicular transporter for GABA, vesicular GABA transporter (VGAT). Among the Orx+ varicosities, 4% of all and 28% of large varicosities contained VGluT2. A similar proportion of the large Orx+ varicosities contained synaptophysin (Syp), a presynaptic marker for synaptic vesicles. Orx+ varicosities also contacted elements immunostained for postsynaptic density protein-95 (PSD)-95, a postsynaptic marker for glutamatergic synapses. We thus conclude that synaptic release of Glu occurs from Orx terminals within the locus coeruleus and can thus be important for the engagement of noradrenergic neurons in stimulating and maintaining arousal.

Effects of brain-derived neurotrophic factor (BDNF) on glial cells and serotonergic neurones during development.

Serotonergic neurones are among the first to develop in the central nervous system. Their survival and maturation is promoted by a variety of factors, including serotonin itself, brain-derived neurotrophic factor (BDNF) and S100beta, an astrocyte-specific Ca(2+) binding protein. Here, we used BDNF-deficient mice and cell cultures of embryonic raphe neurones to determine whether or not BDNF effects on developing serotonergic raphe neurones are influenced by its action on glial cells. In BDNF-/- mice, the number of serotonin-immunoreactive neuronal somata, the amount of the serotonin transporter, the serotonin content in the striatum and the hippocampus, and the content of 5-hydroxyindoleacetic acid in all brain regions analysed were increased. By contrast, reduced immunoreactivity was found for myelin basic protein (MBP) in all brain areas including the raphe and its target region, the hippocampus. Exogenously applied BDNF increased the number of MBP-immunopositive cells in the respective culture systems. The raphe area displayed selectively reduced immunoreactivity for S100beta. Accordingly, S100beta was increased in primary cultures of pure astrocytes by exogenous BDNF. In glia-free neuronal cultures prepared from the embryonic mouse raphe, addition of BDNF supported the survival of serotonergic neurones and increased the number of axon collaterals and primary dendrites. The latter effect was inhibited by the simultaneous addition of S100beta. These results suggest that the presence of BDNF is not a requirement for the survival and maturation of serotonergic neurones in vivo. BDNF is, however, required for the local expression of S100beta and production of MBP. Therefore BDNF might indirectly influence the development of the serotonergic system by stimulating the expression of S100beta in astrocytes and the production MBP in oligodendrocytes.