Altered urinary tetrahydroisoquinoline derivatives in patients with Tourette syndrome: reflection of dopaminergic hyperactivity?

Tetrahydroisoquinolines (TIQs) such as salsolinol (SAL), norsalsolinol (NSAL) and their methylated derivatives N-methyl-norsalsolinol (NMNSAL) and N-methyl-salsolinol (NMSAL), modulate dopaminergic neurotransmission and metabolism in the central nervous system. Dopaminergic neurotransmission is thought to play an important role in the pathophysiology of chronic tic disorders, such as Tourette syndrome (TS). Therefore, the urinary concentrations of these TIQ derivatives were measured in patients with TS and patients with comorbid attention-deficit/hyperactivity disorder (TS + ADHD) compared with controls. Seventeen patients with TS, 12 with TS and ADHD, and 19 age-matched healthy controls with no medication took part in this study. Free levels of NSAL, NMNSAL, SAL, and NMSAL in urine were measured by a two-phase chromatographic approach. Furthermore, individual TIQ concentrations in TS patients were used in receiver-operating characteristics (ROC) curve analysis to examine the diagnostic value. NSAL concentrations were elevated significantly in TS [434.67 ± 55.4 nmol/l (standard error of mean = S.E.M.), two-way ANOVA, p < 0.0001] and TS + ADHD patients [605.18 ± 170.21 nmol/l (S.E.M.), two-way ANOVA, p < 0.0001] compared with controls [107.02 ± 33.18 nmol/l (S.E.M.), two-way ANOVA, p < 0.0001] and NSAL levels in TS + ADHD patients were elevated significantly in comparison with TS patients (two-way ANOVA, p = 0.017). NSAL demonstrated an AUC of 0.93 ± 0.046 (S.E.M) the highest diagnostic value of all metabolites for the diagnosis of TS. Our results suggest a dopaminergic hyperactivity underlying the pathophysiology of TS and ADHD. In addition, NSAL concentrations in urine may be a potential diagnostic biomarker of TS.

Electrocorticographic and neurochemical findings after local cortical valproate application in patients with pharmacoresistant focal epilepsy.

Because oral pharmacological treatment of neocortical focal epilepsy is limited due to common systemic side effects and relatively low drug concentrations reached at the epileptic foci locally, application of antiepileptic agents directly onto the neocortical focus may enhance treatment tolerability and efficacy. We describe the effects of cortically applied sodium valproate (VPA) in two patients with pharmacoresistant neocortical focal epilepsy who were selected for epilepsy surgery after a circumscribed epileptic focus had been determined by invasive presurgical evaluation using subdural electrodes. Local VPA modified epileptic activity as electrocorticographically recorded from the chronic focus in both patients. In addition, VPA induced local increase of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in cortical tissue samples, whereas the excitatory glutamate was possibly decreased. In this clinical pilot study, we could show antiepileptic effects of cortically applied VPA in humans by electrocorticographic and neurochemical parameters.

Neocortical GABA release at high intracellular sodium and low extracellular calcium: an anti-seizure mechanism.

In epilepsy, the GABA and glutamate balance may be disrupted and a transient decrease in extracellular calcium occurs before and during a seizure. Flow Cytometry based fluorescence activated particle sorting experiments quantified synaptosomes from human neocortical tissue, from both epileptic and non-epileptic patients (27.7% vs. 36.9% GABAergic synaptosomes, respectively). Transporter-mediated release of GABA in human and rat neocortical synaptosomes was measured using the superfusion technique for the measurement of endogenous GABA. GABA release was evoked by either a sodium channel activator or a sodium/potassium-ATPase inhibitor when exocytosis was possible or prevented, and when the sodium/calcium exchanger was active or inhibited. The transporter-mediated release of GABA is because of elevated intracellular sodium. A reduction in the extracellular calcium increased this release (in both non-epileptic and epileptic, except Rasmussen encephalitis, synaptosomes). The inverse was seen during calcium doubling. In humans, GABA release was not affected by exocytosis inhibition, that is, it was solely transporter-mediated. However, in rat synaptosomes, an increase in GABA release at zero calcium was only exhibited when the exocytosis was prevented. The absence of calcium amplified the sodium/calcium exchanger activity, leading to elevated intracellular sodium, which, together with the stimulation-evoked intracellular sodium increment, enhanced GABA transporter reversal. Sodium/calcium exchange inhibitors diminished GABA release. Thus, an important seizure-induced extracellular calcium reduction might trigger a transporter- and sodium/calcium exchanger-related anti-seizure mechanism by augmenting transporter-mediated GABA release, a mechanism absent in rats. Uniquely, the additional increase in GABA release because of calcium-withdrawal dwindled during the course of illness in Rasmussen encephalitis. Seizures cause high Na(+) influx through action potentials. A transient decrease in [Ca(2+)]e (seizure condition) increases GABA transporter (GAT)-mediated GABA release because of elevated [Na(+)]i. This amplifies the Sodium-Calcium-Exchanger (NCX) activity, further increasing [Na(+)]i and GABA release. The reduction in [Ca(2+)]e triggers a GAT-NCX related anti-seizure mechanism by augmenting GAT-mediated GABA release. This mechanism, obvious in humans, is absent in rats.

Inositol 1,4,5-trisphosphate receptor type 1 autoantibodies in paraneoplastic and non-paraneoplastic peripheral neuropathy.

BACKGROUND: Recently, we described a novel autoantibody, anti-Sj/ITPR1-IgG, that targets the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) in patients with cerebellar ataxia. However, ITPR1 is expressed not only by Purkinje cells but also in the anterior horn of the spinal cord, in the substantia gelatinosa and in the motor, sensory (including the dorsal root ganglia) and autonomic peripheral nervous system, suggesting that the clinical spectrum associated with autoimmunity to ITPR1 may be broader than initially thought. Here we report on serum autoantibodies to ITPR1 (up to 1:15,000) in three patients with (radiculo)polyneuropathy, which in two cases was associated with cancer (ITPR1-expressing adenocarcinoma of the lung, multiple myeloma), suggesting a paraneoplastic aetiology. METHODS: Serological and other immunological studies, and retrospective analysis of patient records. RESULTS: The clinical findings comprised motor, sensory (including severe pain) and autonomic symptoms. While one patient presented with subacute symptoms mimicking Guillain-Barré syndrome (GBS), the symptoms progressed slowly in two other patients. Electrophysiology revealed delayed F waves; a decrease in motor and sensory action potentials and conduction velocities; delayed motor latencies; signs of denervation, indicating sensorimotor radiculopolyneuropathy of the mixed type; and no conduction blocks. ITPR1-IgG belonged to the complement-activating IgG1 subclass in the severely affected patient but exclusively to the IgG2 subclass in the two more mildly affected patients. Cerebrospinal fluid ITPR1-IgG was found to be of predominantly extrathecal origin. A 3H-thymidine-based proliferation assay confirmed the presence of ITPR1-reactive lymphocytes among peripheral blood mononuclear cells (PBMCs). Immunophenotypic profiling of PBMCs protein demonstrated predominant proliferation of B cells, CD4 T cells and CD8 memory T cells following stimulation with purified ITPR1 protein. Patient ITPR1-IgG bound both to peripheral nervous tissue and to lung tumour tissue. A nerve biopsy showed lymphocyte infiltration (including cytotoxic CD8 cells), oedema, marked axonal loss and myelin-positive macrophages, indicating florid inflammation. ITPR1-IgG serum titres declined following tumour removal, paralleled by clinical stabilization. CONCLUSIONS: Our findings expand the spectrum of clinical syndromes associated with ITPR1-IgG and suggest that autoimmunity to ITPR1 may underlie peripheral nervous system diseases (including GBS) in some patients and may be of paraneoplastic origin in a subset of cases.

Nasal administration of leptin dose-dependently increases dopamine and serotonin outflow in the rat nucleus accumbens.

Leptin is an anorexigenic hormone that acts via its receptor (LepR) to regulate the hypothalamic arcuate nucleus circuitry to mediate energy homeostasis and feeding behavior. Moreover, leptin decreases the reward value of natural and artificial rewards, and low levels of circulating leptin have been implicated in several mood disorders linking leptin to the mesolimbic system. Therefore, the purpose of this study was to assess whether and to what extent an acute intranasal application of leptin is able to modulate monoamine neurotransmitters in the nucleus accumbens (NAc). Microdialysis experiments were carried out in freely moving Wistar rats and in LepR-deficient Zucker rats (LepRfa/fa). Samples were analysed for the levels of dopamine (DA), serotonin (5-HT), and their metabolites using high-performance liquid chromatography with electrochemical detection. We show that in Wistar rats, nasal application of leptin dose-dependently increased extracellular DA and 5-HT levels in the NAc. By contrast, in the LepRfa/fa rats, nasal application of 0.12 mg/kg leptin failed to increase levels of either DA or 5-HT, but their metabolites (DOPAC and HIAA, respectively) were significantly decreased. In addition, leptin interaction with the melanocortin system was tested. Nasal co-administration of leptin and the melanocortin receptor antagonist, SHU9119, completely abolished the leptin-induced increase of both DA and 5-HT outflow in the NAc. These results indicate a marked leptin effect on the basal ganglia-related reward system involving melanocortin receptors.

Exploring Network Formation of Tough and Biocompatible Thiol-yne Based Photopolymers.

This work deals with the in-depth investigation of thiol-yne based network formation and its effect on thermomechanical properties and impact strength. The results show that the bifunctional alkyne monomer di(but-1-yne-4-yl)carbonate (DBC) provides significantly lower cytotoxicity than the comparable acrylate, 1,4-butanediol diacrylate (BDA). Real-time near infrared photorheology measurements reveal that gel formation is shifted to higher conversions for DBC/thiol resins leading to lower shrinkage stress and higher overall monomer conversion than BDA. Glass transition temperature (Tg ), shrinkage stress, as well as network density determined by double quantum solid state NMR, increase proportionally with the thiol functionality. Most importantly, highly cross-linked DBC/dipentaerythritol hexa(3-mercaptopropionate) networks (Tg ≈ 61 °C) provide a 5.3 times higher impact strength than BDA, which is explained by the unique network homogeneity of thiol-yne photopolymers.

High-frequency electrical stimulation suppresses cholinergic accumbens interneurons in acute rat brain slices through GABA(B) receptors.

The nucleus accumbens is selected as a surgical target in deep brain stimulation for treating refractory obsessive-compulsive disorder (OCD). One of the therapeutic benefits of this procedure is that the abnormal hyper-functioning prefrontal cortex of patients with OCD is restored during stimulation. One hypothesis regarding the mechanism of deep brain stimulation is that the neuronal electrophysiological properties are directly altered by electrical stimulation; another hypothesis assumes that the stimulation induces selective neuron transmitter release, such as γ-aminobutyric acid (GABA). In this study, we used multi-electrode arrays with electrode size of 40 × 40 µm to record electrophysiological signals from the large nucleus accumbens neurons in acute rat brain slices while applying electrical stimulation simultaneously. We revealed that high-frequency stimulation (HFS, 140 Hz) suppressed the spontaneous neuronal firing rate significantly, whereas low-frequency stimulation (LFS, 10 Hz) did not. Both HFS and LFS have no effect on neuronal firing pattern or on neuronal oscillation synchrony. GABAB receptor antagonism reversed the HFS-provoked neuronal inhibition, whereas GABAA receptor blockade failed to affect it. The recorded neurons were pharmacologically identified to be cholinergic interneurons. We propose that HFS has a direct suppressive effect on the identified accumbal acetylcholine (ACh) interneurons by enhancing GABA release in the stimulated region. Potentially, suppressed ACh interneurons decrease the disinhibiting function of medium-sized spiny neurons in the striato-thalamo-cortical circuit. This finding might give an indication of the mechanism of the therapeutic effect of HFS in nucleus accumbens on restoring the abnormal hyperactive prefrontal cortex status in OCD.

Transmitter self-regulation by extracellular glutamate in fresh human cortical slices.

Glutamate is thought to be the most important excitatory neurotransmitter in the CNS, while glutamine predominantly serves as a precursor and metabolite in the glutamate-glutamine cycle. To verify the interaction between intrinsic extracellular glutamate, y-aminobutyric acid (GABA) levels and glial glutamine outflow in human tissue, fresh brain slices from human frontal cortex were incubated in superfusion chambers in vitro. Human neocortical tissue was obtained during surgical treatment of subcortical brain tumors. For superfusion experiments, the white matter was separated and discarded from the gray matter, which finally contained all six neocortical layers. Outflows of endogenous glutamate, GABA and glutamine were established after a 40-min washout period and amounts were simultaneously quantified after two-phase derivatization by high-performance liquid chromatography with electrochemical detection. Under basal conditions, amounts of glutamate could be found 20-fold in comparison to the inhibitory neurotransmitter GABA, whereas this excitatory predominance markedly declined after veratridine-induced activation. The basal glutamate:glutamine ratio of extracellular levels was approximately 1:2. Blockade or activation of the voltage-gated sodium channel by tetrodotoxin or veratridine significantly modulated glutamate levels, but the glutamate:glutamine ratio was nearly constant with 1:2. When the EAAT blocker TBOA was employed, glutamine remained nearly unchanged whereas glutamate significantly enhanced. These results led us to suggest that glutamine release through glial SN1 is related to EAAT activity that can be modulated by intrinsic extracellular glutamate in human cortical slices.

Persistent cognitive impairment associated with cerebrospinal fluid anti-SARS-CoV-2 antibodies six months after mild COVID-19.

Neurological long-term sequelae are increasingly considered an important challenge in the recent COVID-19 pandemic. However, most evidence for neurological symptoms after SARS-CoV-2 infection and central nervous system invasion of the virus stems from individuals severely affected in the acute phase of the disease. Here, we report long-lasting cognitive impairment along with persistent cerebrospinal fluid anti-SARS-CoV-2 antibodies in a female patient with unremarkable standard examination 6 months after mild COVID-19, supporting the implementation of neuropsychological testing and specific cerebrospinal fluid investigation also in patients with a relatively mild acute disease phase.

Electrical high frequency stimulation of the nucleus accumbens shell does not modulate depressive-like behavior in rats.

Deep brain stimulation (DBS) is an effective tool for treatment-resistant depression, though it is still unclear which brain area to target in order to get robust results. While research suggests that the nucleus accumbens (NAc) plays an important role in depression, studies using NAc DBS to improve depressive behavior have not been able to fully explain underlying molecular mechanisms. We therefore used unilateral high frequency stimulation of the NAc shell in rats to verify its effectiveness in treating depression and study involved neurotransmitter systems. Animals underwent social isolation and food deprivation to induce depressive-like symptoms, and performed the forced swim test (FST) to see possible changes in depressive behavior due to NAc shell stimulation. Neurotransmitter levels were measured using in-vivo microdialysis to detect DBS-induced changes. Non-treated control rats showed no significant difference between swimming and floating during FST, verifying that our depression model induced depressive-like symptoms in rats. Furthermore, stimulated and sham-operated animals showed a significant increase in mobility during FST compared to control rats, suggesting an improvement of depressive-like symptoms. However, stimulated rats did not differ from sham-operated rats in their behavior during FST, nor did neurotransmitter levels significantly changed in stimulated rats compared to control rats. Our data suggest that NAc shell stimulation did not alter depressive behavior in rats and had no effects on the molecular level. However, depressive behavior was positively altered when stimulation electrode and microdialysis probe were inserted simultaneously into the NAc shell, causing significant tissue damage and therefore possibly altering the glutamatergic system.

Endogenous tetrahydroisoquinolines associated with Parkinson's disease mimic the feedback inhibition of tyrosine hydroxylase by catecholamines.

N-methyl-norsalsolinol and related tetrahydroisoquinolines accumulate in the nigrostriatal system of the human brain and are increased in the cerebrospinal fluid of patients with Parkinson's disease. We show here that 6,7-dihydroxylated tetrahydroisoquinolines such as N-methyl-norsalsolinol inhibit tyrosine hydroxylase, the key enzyme in dopamine synthesis, by imitating the mechanisms of catecholamine feedback regulation. Docked into a model of the enzyme's active site, 6,7-dihydroxylated tetrahydroisoquinolines were ligated directly to the iron in the catalytic center, occupying the same position as the catecholamine inhibitor dopamine. In this position, the ligands competed with the essential tetrahydropterin cofactor for access to the active site. Electron paramagnetic resonance spectroscopy revealed that, like dopamine, 6,7-dihydroxylated tetrahydroisoquinolines rapidly convert the catalytic iron to a ferric (inactive) state. Catecholamine binding increases the thermal stability of tyrosine hydroxylase and improves its resistance to proteolysis. We observed a similar effect after incubation with N-methyl-norsalsolinol or norsalsolinol. Following an initial rapid decline in tyrosine hydroxylation, the residual activity remained stable for 5 h at 37 degrees C. Phosphorylation by protein kinase A facilitates the release of bound catecholamines and is the most prominent mechanism of tyrosine hydroxylase reactivation. Protein kinase A also fully restored enzyme activity after incubation with N-methyl-norsalsolinol, demonstrating that tyrosine hydroxylase inhibition by 6,7-dihydroxylated tetrahydroisoquinolines mimics all essential aspects of catecholamine end-product regulation. Increased levels of N-methyl-norsalsolinol and related tetrahydroisoquinolines are therefore likely to accelerate dopamine depletion in Parkinson's disease.

Unmet digital health service needs in dermatology patients.

BACKGROUND: Digital health services are rapidly gaining acceptance in healthcare systems. Dermatology as an image-centric specialty is particularly well suited for telemedical services. However, dermatology patients' demands of electronic services remain largely unexplored. METHODS: This study investigated patients' views in primary, secondary, and tertiary referral centers. In August 2017, 841 questionnaires were filled in by dermatology patients. RESULTS: 76.34% expressed interest in using digital healthcare services as part of medical consultations. 84.41% of all patients would complete their initial registration form electronically. Fewer patients were comfortable with sending pictures of skin changes to their doctors using email (40.89%) or mobile health applications (40.61%). Specific interest was indicated for arranging appointments online (90.80%) and electronically-placed prescriptions (76.56%), rather than online learning videos (42.03%), and actual online consultations (34.53%). 65.37% of patients would pay for online consultations themselves. CONCLUSIONS: Taken together, interest in electronic health services is high in dermatology patients. Our data suggest that readily understandable electronic services such as online-arranged appointments and electronic prescriptions are of higher interest to patients than the current type of online consultations. Therefore, the full potential of teledermatology still remains to be tapped by newer, more attractive forms of services closely adapted to patients' demands.

K(ATP)-dependent neurotransmitter release in the neuronal network of the rat caudate nucleus.

K(ATP) channels can couple the bioenergetic metabolism of the cell to membrane excitability. Here, we show gamma-aminobutyric acid (GABA) mediated inhibition of dopamine outflow from slices of the rat caudate nucleus that is regulated by extracellular glucose via high- and low-affinity K(ATP) channels. During glucose reduction, a biphasic dopamine effect could be observed with first a dopamine increase followed by a decline at low glucose concentrations. Both phases were inhibited by glibenclamide. Pinacidil decreased DA outflow without an effect of glucose reduction implying an overall activation of K(ATP) channels. The first phase with dopamine increase was related to reduced GABAergic activity and could be blocked by bicuculline. Our results may be explained by different types of K(ATP) channels with low affinity of ATP and glibenclamide on inhibitory GABAergic and high-affinity on excitatory DAergic neurons. This led us to suggest a biological principle through which neuronal networks are functioning.

Tetrahydroisoquinoline derivatives: a new perspective on monoaminergic dysfunction in children with ADHD?

BACKGROUND: The dopamine-derived tetrahydroisoquinolines (TIQ) synthesized endogeneously from aldehydes and catecholamines have shown to modulate neurotransmission, central metabolism and motor activity. Converging evidence has implicated abnormalities of the dopamine metabolism to the pathophysiology of Attention-Deficit/Hyperactivity Disorder (ADHD). Therefore, four TIQ derivatives involved in central dopamine metabolism (salsolinol, N-methyl-salsolinol, norsalsolinol, N-methyl-norsalsolinol) have been analyzed for the first time in children and adolescents with ADHD and healthy controls. METHODS: 42 children and adolescents with ADHD and 24 controls from three sites participated in this pilot study. Free and bound amounts of salsolinol, N-methyl-salsolinol, norsalsolinol, N-methyl-norsalsolinol have been analyzed in urine. RESULTS: In the ADHD group, free and total amounts of the four TIQ derivatives in urine were significantly higher compared to urine levels of healthy controls. For N-methyl-salsolinolfree, most of the ADHD patients were identified correctly with a sensitivity of 92.5% (specificity 94.4%). CONCLUSION: Urine levels of salsolinol, N-methyl-salsolinol, norsalsolinol and N-methyl-norsalsolinol are elevated in children and adolescents with ADHD and point to a new perspective on catecholaminergic dysfunction in ADHD. However, replication and extension of this pilot study would progress this innovative and promising field.

Inkjet Printing of Soft, Stretchable Optical Waveguides through the Photopolymerization of High-Profile Linear Patterns.

Optical waveguides have been fabricated via photopolymerization of stable, inkjet-printed patterns. In order to obtain high-profile lines, the properties of both the ink and the substrate were adjusted. We prove that suitable patterns, with contact angles close to 90°, can be printed by using not fully cured, "sticky" PDMS as a substrate. In addition, we propose a simple sliding-drop experiment to show the crucial difference in how the ink dewets the "sticky" and the fully cured substrate, which is otherwise difficult to demonstrate. The light attenuation vs strain curve of the obtained waveguides was determined experimentally and was found to be almost linear within the measured strain range.

Modulation of a neuronal network by electrical high frequency stimulation in striatal slices of the rat in vitro.

The effects of the GABA(A) receptor antagonist bicuculline, the D2-like receptor antagonist sulpiride and the D1-like receptor antagonist SCH-23390 on the electrical high frequency stimulation (HFS)-evoked gamma-aminobutyric acid (GABA) and dopamine (DA) release were measured from slices of the rat striatum by means of HPLC method with electrochemical detection. HFS with 130Hz stimulated veratridine-activated GABAergic neurons resulting in an increased GABA outflow while DA outflow decreased. In the presence of the GABA(A) receptor antagonist bicuculline extracellular GABA and DA outflow were enhanced. When the competitive dopamine D2-like receptor antagonist S-(-)-sulpiride was added to incubation medium, the HFS-evoked stimulatory effect on GABA outflow declined to values found after veratridine (1microM) without HFS. After co-incubation of sulpiride and the competitive D1-like receptor antagonist R-(+)-SCH-23390, the effect of sulpiride on HFS plus veratridine-evoked GABA outflow was completely reversed. Neither sulpiride nor SCH-23390 had any influence on the effect of HFS on veratridine-induced DA outflow. No effect of HFS on glutamate outflow was observed in all experiments. These results led us to suggest that in our model HFS primarily affects GABAergic neurons. These neurons are embedded in a neuronal network with a GABA-dopamine circuit, and thus, HFS interacts with a neuronal network, not only with one neurotransmitter system or one neuron population.

Mechanical Properties of Composites Used in High-Voltage Applications.

Materials used in high voltage applications have to meet a lot of regulations for their safety and functional usage during their lifetime. For high voltage applications the electrical properties are the most relevant designing criteria. However, the mechanical properties of such materials have rarely been considered for application dimensioning over the last decades. This article gives an overview of composite materials used in high voltage applications and some basic mechanical and thermo-mechanical characterization methods of such materials, including a discussion of influences on practically used epoxy based thermosets.

Data on synthesis and thermo-mechanical properties of stimuli-responsive rubber materials bearing pendant anthracene groups.

The photo-reversible [4πs+4πs] cycloaddition reaction of pendant anthracene moieties represents a convenient strategy to impart wavelength dependent properties into hydrogenated carboxylated nitrile butadiene rubber (HXNBR) networks. The present article provides the 1H NMR data on the reaction kinetics of the side chain functionalization of HXNBR. 2-(Anthracene-9-yl)oxirane with reactive epoxy groups is covalently attached to the polymer side chain of HXNBR via ring opening reaction between the epoxy and the carboxylic groups. Along with the identification, 1H NMR data on the quantification of the attached functional groups are shown in dependence on reaction time and concentration of 2-(anthracene-9-yl)oxirane. Changes in the modification yield are reflected in the mechanical properties and DMA data of photo-responsive elastomers are illustrated in dependence on the number of attached anthracene groups. DMA curves over repeated cycles of UV induced crosslinking (λ>300 nm) and UV induced cleavage (λ=254 nm) are further depicted, demonstrating the photo-reversibility of the thermo-mechanical properties. Interpretation and discussion of the data are provided in "Design and application of photo-reversible elastomer networks by using the [4πs+4πs] cycloaddition reaction of pendant anthracene groups" (Manhart et al., 2016) [1].

Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts.

Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at all time points.

Electrical high frequency stimulation modulates GABAergic activity in the nucleus accumbens of freely moving rats.

Deep brain stimulation (DBS) of the nucleus accumbens (NAc) is an effective treatment option for those affected by obsessive compulsive disorder, who do not respond to pharmacological treatment strategies. Yet, little is known about the mechanism by which DBS achieve its therapeutic effects. Previous studies have shown an increase in GABA levels due to high frequency stimulation (HFS) in the rat caudate putamen. Here, the effect of high frequency stimulation in the nucleus accumbens of conscious and freely moving rats was characterized using unilateral but simultaneous microdialysis and HFS with a frequency of 124 Hz and 0.5 mA current. Extracellular levels of neurotransmitters - GABA, glutamate, dopamine, serotonin and their metabolites were quantified by means of HPLC with electrochemical detection. Basal levels of GABA were significantly increased in animals of the stimulation group compared to the control group without HFS. The levels of other neurotransmitters were unaffected. The influence of NMDA receptor antagonist, memantine (5 mg/kg) on the effect of HFS was investigated by subcutaneous administration of memantine on the day of the experiment. Memantine (without stimulation) enhanced basal GABA and dopamine levels. However, under the influence of both memantine and HFS, GABA levels were not affected by HFS whereas dopamine levels decreased during the stimulation period. The results of our study demonstrate that HFS in the nucleus accumbens of freely moving rats induces selective increase in GABA outflow and show a possible involvement of NMDA receptors in the mechanistic action of HFS.