The beta-isoform of neuronal nitric oxide synthase (nNOS) lacking the PDZ domain is localized at the sarcolemma

In skeletal muscles, the expression of neuronal NO synthase (nNOS) isoforms is uncharacterized at the protein level. We therefore conducted epitope mapping with anti-peptide-antibodies. Antibodies specific for the nNOS N-terminus recognized the 160-kDa alpha-isoform. In contrast, antibodies against the middle portion or the C-terminus of nNOS bound additionally to the truncated 140-kDa beta-isoform which lacks the PDZ-domain present in the alpha-isoform. All nNOS immunohistochemical reactivity was confined to the sarcolemma. Consistently, immunoblotting disclosed both nNOS-isoforms to be co-enriched in the membrane-associated fractions. The beta-isoform was co-immunoprecipitated with alpha-isoform antibodies in muscle extracts indicating an association of both nNOS-isoforms to direct the beta-variant to the sarcolemma.

Exercise-induced angiogenesis correlates with the up-regulated expression of neuronal nitric oxide synthase (nNOS) in human skeletal muscle

The contribution of neuronal nitric oxide synthase (nNOS) to angiogenesis in human skeletal muscle after endurance exercise is controversially discussed. We therefore ascertained whether the expression of nNOS is associated with the capillary density in biopsies of the vastus lateralis (VL) muscle that had been derived from 10 sedentary male subjects before and after moderate training (four 30-min weekly jogging sessions for 6 months, with a heart-rate corresponding to 75% VO(2)max). In these biopsies, nNOS was predominantly expressed as alpha-isoform with exon-mu and to a lesser extent without exon-mu, as determined by RT-PCR. The mRNA levels of nNOS were quantified by real-time PCR and related to the capillary-to-fibre ratio and the numerical density of capillaries specified by light microscopy. If the VL biopsies of all subjects were co-analysed, mRNA levels of nNOS were non-significantly elevated after training (+34%; P > 0.05). However, only five of the ten subjects exhibited significant (P ≤ 0.05) elevations in the capillary-to-fibre ratio (+25%) and the numerical density of capillaries (+21%) and were thus undergoing angiogenesis. If the VL biopsies of these five subjects alone were evaluated, the mRNA levels of nNOS were significantly up-regulated (+128%; P ≤ 0.05) and correlated positively (r = 0.8; P ≤ 0.01) to angiogenesis. Accordingly, nNOS protein expression in VL biopsies quantified by immunoblotting was significantly increased (+82%; P ≤ 0.05) only in those subjects that underwent angiogenesis. In conclusion, the expression of nNOS at mRNA and protein levels was statistically linked to capillarity after exercise suggesting that nNOS is involved in the angiogenic response to training in human skeletal muscle.

Syntrophin mutation associated with long QT syndrome through activation of the nNOS-SCN5A macromolecular complex.

Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for approximately 75-80% of cases (LQT1-11). Direct sequencing of SNTA1, the gene encoding alpha1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTS-susceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane Ca-ATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused S-nitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.

Phenotype of capillaries in skeletal muscle of nNOS-knockout mice

Because neuronal nitric oxide synthase (nNOS) has a well-known impact on arteriolar blood flow in skeletal muscle, we compared the ultrastructure and the hemodynamics of/in the ensuing capillaries in the extensor digitorum longus (EDL) muscle of male nNOS-knockout (KO) mice and wild-type (WT) littermates. The capillary-to-fiber (C/F) ratio (-9.1%) was lower (P ≤ 0.05) in the nNOS-KO mice than in the WT mice, whereas the mean cross-sectional fiber area (-7.8%) and the capillary density (-3.1%) varied only nonsignificantly (P > 0.05). Morphometrical estimation of the area occupied by the capillaries as well as the volume and surface densities of the subcellular compartments differed nonsignificantly (P > 0.05) between the two strains. Intravital microscopy revealed neither the capillary diameter (+3% in nNOS-KO mice vs. WT mice) nor the mean velocity of red blood cells in EDL muscle (+25% in nNOS-KO mice vs. WT mice) to significantly vary (P > 0.05) between the two strains. The calculated shear stress in the capillaries was likewise nonsignificantly different (3.8 ± 2.2 dyn/cm² in nNOS-KO mice and 2.1 ± 2.2 dyn/cm² in WT mice; P > 0.05). The mRNA levels of vascular endothelial growth factor (VEGF)-A were lower in the EDL muscle of nNOS-KO mice than in the WT littermates (-37%; P ≤ 0.05), whereas mRNA levels of VEGF receptor-2 (VEGFR-2) (-11%), hypoxia inducible factor-1α (+9%), fibroblast growth factor-2 (-14%), and thrombospondin-1 (-10%) differed nonsignificantly (P > 0.05). Our findings support the contention that VEGF-A mRNA expression and C/F-ratio but not the ultrastructure or the hemodynamics of/in capillaries in skeletal muscle at basal conditions depend on the expression of nNOS.

Empathie in der Psychotherapie. Neuronale Grundlagen und Implikationen für die Praxis

Empathie ist ein Begriff, der in einer Vielzahl von Kontexten verwendet wird und daher auch sehr unterschiedlich verstanden werden kann. So wird in dieser Arbeit eine Definition von Empathie gesucht, die Empathie von ähnlichen Konstrukten abgrenzt und auch in der psychotherapeutischen Praxis angewandt werden kann. In der Psychotherapie spielt die Empathie des Therapeuten eine wichtige Rolle für den Verlauf und die Ergebnisse der Therapie. Seit dem Aufkommen neuer Forschungsmethoden mit Hilfe bildgebender Verfahren lassen sich die neuronalen Korrelate der Empathie genauer untersuchen. Dies lässt auch neue Erkenntnisse in Bezug auf die psychotherapeutische Praxis zu. Daher sollen hier einige neurowissenschaftliche Forschungsergebnisse aufgezeigt werden, woraus Implikationen für die psychotherapeutische Praxis abgeleitet werden. Ausserdem werden Modulationen und Grenzen der therapeutischen Empathie und Schwerpunkte eines möglichen Empathie-Trainings anhand ausgewählter wissenschaftlicher Literatur aufgezeigt.

Creatine and neurotrophin-4/5 promote survival of nitric oxide synthase-expressing interneurons in striatal cultures

Nitric oxide (NO) mediates a variety of physiological functions in the central nervous system and acts as an important developmental regulator. Striatal interneurons expressing neuronal nitric oxide synthase (nNOS) have been described to be relatively spared from the progressive cell loss in Huntington's disease (HD). We have recently shown that creatine, which supports the phosphagen energy system, induces the differentiation of GABAergic cells in cultured striatal tissue. Moreover, neurotrophin-4/5 (NT-4/5) has been found to promote the survival and differentiation of cultured striatal neurons. In the present study, we assessed the effects of creatine and NT-4/5 on nNOS-immunoreactive (-ir) neurons of E14 rat ganglionic eminences grown for 1 week in culture. Chronic administration of creatine [5mM], NT-4/5 [10ng/ml], or a combination of both factors significantly increased numbers of nNOS-ir neurons. NT-4/5 exposure also robustly increased levels of nNOS protein. Interestingly, only NT-4/5 and combined treatment significantly increased general viability but no effects were seen for creatine supplementation alone. In addition, NT-4/5 and combined treatment resulted in a significant larger soma size and number of primary neurites of nNOS-ir neurons while creatine administration alone exerted no effects. Double-immunolabeling studies revealed that all nNOS-ir cells co-localized with GABA. In summary, our findings suggest that creatine and NT-4/5 affect differentiation and/or survival of striatal nNOS-ir GABAergic interneurons. These findings provide novel insights into the biology of developing striatal neurons and highlight the potential of both creatine and NT-4/5 as therapeutics for HD.

Insulin resistance in mice lacking neuronal nitric oxide synthase is related to an alpha-adrenergic mechanism

BACKGROUND: nitric oxide (NO) plays an important role in the regulation of cardiovascular and glucose homeostasis. Mice lacking the gene encoding the neuronal isoform of nitric oxide synthase (nNOS) are insulin-resistant, but the underlying mechanism is unknown. nNOS is expressed in skeletal muscle tissue where it may regulate glucose uptake. Alternatively, nNOS driven NO synthesis may facilitate skeletal muscle perfusion and substrate delivery. Finally, nNOS dependent NO in the central nervous system may facilitate glucose disposal by decreasing sympathetic nerve activity. METHODS: in nNOS null and control mice, we studied whole body glucose uptake and skeletal muscle blood flow during hyperinsulinaemic clamp studies in vivo and glucose uptake in skeletal muscle preparations in vitro. We also examined the effects of alpha-adrenergic blockade (phentolamine) on glucose uptake during the clamp studies. RESULTS: as expected, the glucose infusion rate during clamping was roughly 15 percent lower in nNOS null than in control mice (89 (17) vs 101 (12) [-22 to -2]). Insulin stimulation of muscle blood flow in vivo, and intrinsic muscle glucose uptake in vitro, were comparable in the two groups. Phentolamine, which had no effect in the wild-type mice, normalised the insulin sensitivity in the mice lacking the nNOS gene. CONCLUSIONS: insulin resistance in nNOS null mice was not related to defective insulin stimulation of skeletal muscle perfusion and substrate delivery or insulin signaling in the skeletal muscle cell, but to a sympathetic alpha-adrenergic mechanism.

Differential vulnerability of immature murine neurons to oxygen-glucose deprivation

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6-8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions-oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.

Olfactory bulb interneurons releasing NO exhibit the Reelin receptor ApoEr2 and part of those targeted by NO express Reelin

Nitric oxide (NO) and Reelin both modulate neuronal plasticity in developing and mature synaptic networks. We recently showed a loss of neuronal nitric oxide synthase (nNOS) protein in the olfactory bulb of reeler mutants and advanced the hypothesis that the Reelin and NO signalling pathways may influence each other. We now studied the distribution of NO sensitive guanylyl cyclase (NOsGC), Reelin and its receptor Apolipoprotein E2 (ApoEr2) in the olfactory bulb by multiple fluorescence labelling and tested whether nNOS and ApoEr2 colocalize in this area. We also essayed the protein content of NOsGC in the reeler olfactory bulb and tested whether there are any changes in nNOS and NOsGC protein in other reeler brain areas. Olfactory bulb interneurons expressing ApoEr2 and nNOS are only few in the glomerular layer but represent the large majority of granule cell layer interneurons. Conversely, NOsGC interneurons are rare in the granule cell layer and abundant as periglomerular cells. Reelin containing periglomerular cells almost entirely belong to the NOsGC subset. These data further support the hypothesis of a reciprocal signalling between Reelin/NOsGC and ApoEr2/nNOS expressing neurons to affect olfactory bulb activity. We also show that a significant rise in NOsGC content accompanies the decrease of nNOS protein in the reeler olfactory bulb. The same reciprocal changes present in the cortex/striatum and the hippocampus of reeler mice. Thus, the influence that the deficit of extracellular Reelin seems to exert on nNOS and its receptor is not limited to the olfactory bulb but is a general feature of the reeler brain.

Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase

Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.

Psychotherapie und Neuroökonomie

Es gibt zunehmend Beispiele, die belegen, dass die Neuroökonomie als Kombination von ökonomischer Entscheidungstheorie und Neurowissenschaften einen wichtigen Beitrag zur Psychotherapie-Forschung leisten kann. Die Berührungspunkte der beiden Disziplinen sind vielfältig: • Neuroökonomie benutzt Verhaltensexperimente, die es erlauben, komplexes menschliches Verhalten zu untersuchen. Psychotherapie verändert komplexes menschliches Verhalten. Zur Verbesserung der Diagnostik und der Evaluation von Therapieergebnissen können einfache neuroökonomische Experimente einen wichtigen Beitrag leisten. Die experimentelle Messung von zeitlichen, sozialen und Unsicherheitspräferenzen ist besonders geeignet, psychische Störungen zu charakterisieren. • Neuroökonomie ist eine Wissenschaft der menschlichen Motivation. Das Verständnis von bewussten und unbewussten Motivationsfaktoren erlaubt es Psychotherapeutinnen, die Komplexität und Tiefe der Probleme ihrer Patientinnen zu erfassen. • Neuroökonomie ist eine Sozialwissenschaft. Beziehungsprobleme gehören zu den häufigsten Klagen von Patientinnen mit psychischen Störungen, soziale Stressoren sind wichtige Ursachen psychischer Störungen und die therapeutische Beziehung ist der wichtigste Wirkfaktor der Psychotherapie. Die neuroökonomische Erforschung des Sozialverhaltens kann deshalb die Psychotherapie auf unterschiedlichen Ebenen inspirieren. • Neuroökonomie ist eine Neurowissenschaft. Psychotherapie-Forschung beschäftigt sich zunehmend mit Neuroplastizität, insbesondere mit den Effekten von Psychotherapie auf die Funktion und die Struktur des Gehirns. Der neuroökonomische Forschungsansatz macht es möglich, komplexe neuronale Funktionsstörungen bei psychischen Krankheiten zu identifizieren und ihre Modifikation durch Psychotherapie sichtbar zu machen. • Neuroökonomie ist eine umfassende Wissenschaft des menschlichen Verhaltens. Moderne Psychotherapie hat den Anspruch, psychische Störungen auf dem Hintergrund eines bio-psycho-soziales Krankheitsmodells zu verstehen und zu behandeln. Die Neuroökonomie kann einen Beitrag leisten, psychotherapeutische Krankheitsmodelle wissenschaftlich zu fundieren. Die ökonomische Entscheidungstheorie ermöglicht es, die Wechselwirkungen und Synergien von psychotherapeutischer Arbeit, somatischen Behandlungen und sozialen Rahmenbedingungen abzuschätzen. Folgende Eigenschaften schränken die Anwendbarkeit von neuroökonomischen Ansätzen in der Psychotherapie-Forschung allerdings ein: • Das Präferenz-Konzept geht von einer stabilen Verhaltensprädisposition aus. Wechsel von Präferenzen und stark situationsabhängiges Verhalten kann nur beschränkt modelliert werden. • In den meisten neuroökonomischen Experimenten wird Geld als allgemein gültiger Anreiz verwendet. Diese Methodik erlaubt es nicht, reizspezifisches Verhalten zu untersuchen. • Die Neuroökonomie abstrahiert soziale Beziehungen, um sie wissenschaftlich fassbar zu machen. Gewisse Beziehungsaspekte wie beispielsweise die Rolle von Gestik und Mimik können mit dieser Methodik nicht untersucht werden. • Die klassische ökonomische Entscheidungstheorie ist besonders geeignet, „kalte“, überlegte Entscheidungen zu verstehen. Impulsives und zeitinkonsistentes Verhalten kann mit dieser Theorie nur ungenügend beschrieben werden. Neuroökonomie ist eine junge Wissenschaft mit grossem Entwicklungspotential. Führende theoretische und Experimentalökonomen sind daran, Theorie und Forschungsmethodik zu erweitern, um situations- und reizspezifische Faktoren besser zu berücksichtigen und das „heisse“ Ende des Spektrums von Entscheidungsfindungen besser zu verstehen.

The canine neuronal ceroid-lipofuscinosis: a review.

The present article gives a survey over the current scientific knowledge of the canine neuronal ceroid-lipofuscinosis (NCL). NCL is a heterogenous group of lysosomal storage diseases in humans and animals. In consequence of a gene mutation, there is an accumulation of ceroid-lipofuscin in neurons, cells of the retina and the skin and other cells. The stored ceroid-lipofuscin in neurons leads to an impaired cell function and subsequently to cell death. Recently, the underlying genetic defect was discovered in several dog breeds. Genetic testing permits an ante mortem diagnosis of the disease, which up to now was only possible with a positive biopsy result. Another advantage is the identification of carrier animals to eliminate the deleterious alleles.

Funktionalität peripherer Wahrnehmung bei Trackingaufgaben

Schlüsselwörter: Multiple-Object-Tracking, Sakkadenlatenz, Erkennungsleistung Einleitung Beim Multiple-Object-Tracking müssen mehrere, sich bewegende Zielobjekte visuell ver-folgt werden. Dabei scheint es vorteilhaft zu sein, den Blick zwischen den Zielobjekten zu verankern, um Bewegungsinformationen peripher wahrzunehmen (Fehd & Seiffert, 2010). Nach Prüfung dieser Annahme (Experiment 1) wurde getestet, wie gut und schnell auf Bewegungs- und Formveränderungen der Zielobjekte reagiert werden kann (Experiment 2), um die Funktionalität der peripheren Wahrnehmung zu überprüfen Methode 14 Teilnehmer hatten die Aufgabe, zum Ende eines Einzelversuchs 4 aus 10 Vierecken wiederzuerkennen, die sich linear für 6 s in einem projizierten Quadrat bewegten. Dabei wurden 3 Geschwindigkeiten (6, 9 und 12°/s) in 9 Blöcken à 15 Versuchen präsentiert, um die Ergebnisse von Fehd und Seiffert (2010) zu replizieren. In Experiment 2 sollten Teilnehmer auf das Anhalten eines Targets oder dessen Formveränderung zur Raute (Manipulation: 0.5 s) mit Knopfdruck reagieren, bei ausbleibender Veränderung hinge-gen die 4 Zielobjekte wiedererkennen (3 Bedingungen in 10 Blöcken à 12 Versuchen). Erwartet wurde, dass Bewegungsveränderungen häufiger und schneller erkannt werden. Ergebnisse Experiment 1 ergab einen signifikanten Haupteffekt für Geschwindigkeit, F(2,26) = 62.66, p < .01, ηp2 = .83, mit höchsten Richtigkeiten bei 6°/s (58%). Ein Haupteffekt für Blickort, F(2,26) = 76.40, p < .01, ηp2 = .85, zeigt, dass der Blick unabhängig von der Geschwindig-keit länger auf dem Centroid war als auf Targets und Distraktoren. Aufgrund der höchs-ten Richtigkeiten bei 6°/s wurde diese Geschwindigkeit in Experiment 2 eingesetzt und festgestellt, dass Bewegungsveränderungen häufiger erkannt werden (83 %) als Form-veränderungen (59 %), F(1,10) = 17.20, p < .01, ηp2 = .63. Unterschiede in Sakkadenla-tenzen, F(1,10) = 6.73, p = .03, ηp2 = .40, deuten auf eine periphere Wahrnehmung der Bewegungsveränderungen hin. Experiment 3 wird zeigen, ob Sakkaden das Monitoring stören. Diskussion Die periphere Wahrnehmung scheint immer dann funktional zu sein, wenn mehrere, für eine Aufgabe relevante Objekte gleichzeitig verfolgt werden müssen und wenn Verände-rungen, besonders der Bewegung, schnell erkannt werden müssen. Weitere Untersu-chungen sollen zeigen, ob diese Funktionalität der peripheren Wahrnehmung auch im Sport (z.B. beim gleichzeitigen Verfolgen mehrerer Gegenspieler) erkannt werden kann. Literatur Fehd, H. M. & Seiffert, A. E. (2010). Looking at the center of the targets helps multiple object tracking. Journal of Vision, 10, 1–13.