Age-dependent effects of the ß3 adrenoceptor agonist CL316,243 on human and rat detrusor muscle strips.

Motility of detrusor smooth muscle includes adrenergic relaxation and cholinergic contraction. Since the latter may be deregulated in overactive bladder (OAB) pathophysiology, anticholinergics are the standard therapy but occasionally less tolerated due to side effects such as dry mouth and constipation. ß3 adrenoceptor agonists also alleviate OAB symptoms by relaxing the detrusor muscle. Their age dependence, however, is far from understood. To address this issue, we induced contractions with KCl (60 mM) and carbachol (from 10 nM to 100 µM) in the presence of the ß3 adrenoceptor agonist CL316,243 (from 0.1 to 10 µM) in both human and rat muscle strips. Our results confirmed that both contractions were attenuated by ß3 adrenoceptor activation in both species, but with differing age dependence. In humans, specimens from mid-life subjects showed a significantly more pronounced effect of CL316,243 in attenuating carbachol-induced contractions than those from aged subjects (Cohen's d of maximal attenuation: 1.82 in mid-life versus 0.13 in aged) without altering EC50. Conversely, attenuation of KCl responses by CL316,243 increased during ageing (Spearman correlation coefficient = -0.584, P<0.01). In rats, both KCl- and carbachol-induced contractions were significantly more attenuated by CL316,243 in samples from adolescent as compared to aged samples. Immunohistochemistry in human detrusor sections proved ß3 adrenoreceptor abundance to remain unaltered during ageing. In conclusion, our findings suggest differential age-dependent changes in human ß3 adrenoceptor-dependent attenuation of detrusor contraction in terms of electromechanical versus pharmacomechanical coupling; they may help understand the differential responsiveness of OAB patients to ß3 agents.

Functional changes in neuronal circuits due to antibody-driven autoimmune response.

Autoimmune-mediated encephalitis syndromes are increasingly being recognized as important clinical entities. They need to be thought of as differential diagnosis in any patient presenting with fast-onset psychosis or psychiatric problems, memory deficits or other cognitive problems, including aphasias, as well as seizures or motor automatisms, but also rigidity, paresis, ataxia or dystonic / parkinsonian symptoms. Diagnosis including imaging and CSF search for antibodies needs to be fast, as progression of these inflammatory processes is often causing scarring of brain tissue, with hypergliosis and atrophy. As these symptoms show, the autoantibodies present in these cases appear to act within the CNS. Several of such antibodies have by now been identified such as IgG directed against NMDA-receptors, AMPA receptors, GABAA and GABAB receptors, and voltage gated potassium channels and proteins of the potassium channel complex (i.e. LGI1 and CASPR2). These are neuropil / surface antigens where antibody interaction can well be envisaged to cause dysfunction of the target protein, including internalization. Others, such as antibodies directed against GAD65 (an intracellular enzyme responsible for GABA-synthesis from glutamate), are discussed to constitute epiphenomena, but not causal agents in disease progression. This review will focus on the current knowledge of antibody interaction mechanisms, especially discussing cellular excitability changes and synaptic interactions in hippocampal and other brain networks. One challenge in this context is to find viable hypotheses for the emergence of both, hyperexcitability and seizures, and presumably reduced synaptic plasticity and underlying cognitive dysfunction.

[Reported and measured olfactory and gustatory findings in a long-COVID cohort]. / Berichtetes und gemessenes Riech- und Schmeckvermögen in einer Long-COVID-Kohorte.

INTRODUCTION: Disorders of the sense of smell and taste are often complained as a long-COVID symptom. In a special long-COVID consultation (ENT-LCS), we wanted to figure out how large the proportion of pathological olfactory or gustatory diagnoses actually is in this cohort. METHODS: 48 adult patients who visited the ENT-LCS because of their own suffering were asked about their history, rated their ability to smell (SER) and taste (SES) with school grades and completed the extended Sniffin' Sticks test and the 3-drop test as a taste test. Diagnoses were made from the SDI smell score and the total taste score using normative values. Correlations were calculated between the measured scores and the self-assessment and between SER and SES. RESULTS: Pathological chemosensory diagnoses were present in 90%. Pathological olfactory diagnoses were twice as common as gustatory ones. No pathological diagnosis could be verified in 10% of the cohort. Anosmia-ageusia syndrome was diagnosed in six patients. SER correlated strongly with SDI. SES correlated moderately with SScore-total. SER correlated strongly with SES. CONCLUSIONS: There is a risk of smell-taste confusion when interpreting reported chemosensory symptoms. This is another reason why reference is made to the importance of quantifying the chemical senses with validated tests.

Modification of the Langendorff system of the isolated beating heart for experimental radiotherapy at a synchrotron: 4000†Gy in a heart beat.

Microbeam radiotherapy could help to cure malignant tumours which are currently still considered therapy-resistant. With an irradiation target in the thoracic cavity, the heart would be one of the most important organs at risk. To assess the acute adverse effects of microbeam irradiation in the heart, a powerful ex vivo tool was created by combining the Langendorff model of the isolated beating mammalian heart with X-Tream dosimetry. In a first pilot experiment conducted at the Biomedical and Imaging Beamline of the Australian Synchrotron, the system was tested at a microbeam peak dose approximately ten times higher than the anticipated future microbeam irradiation treatment doses. The entire heart was irradiated with a dose of 4000 Gy at a dose rate of >6000 Gy s-1, using an array of 50 µm-wide microbeams spaced at a centre-to-centre distance of 400 µm. Although temporary arrhythmias were seen, they reverted spontaneously to a stable rhythm and no cardiac arrest occurred. This amazing preservation of cardiac function is promising for future therapeutic approaches.

High prevalence of long-term olfactory dysfunction confirmed by olfactory testing after a community COVID-19 outbreak.

PURPOSE: The prevalence of long-term olfactory and gustatory dysfunction in participants suffering from sudden chemosensory loss due to coronavirus disease 2019 (COVID-19) is unknown. Furthermore, evaluations of the reliability of participants' self-reporting of olfactory function (SOF) and gustatory function (SGF) using extended objective psychophysical testing are missing. METHODS: In this population-based cohort study in a PCR-tested community in Thuringia, Germany, olfactory function was extensively examined 4 months after a COVID-19 outbreak using the "Sniffin Sticks" test battery to determine the TDIa score, i.e., the sum of results obtained for threshold, discrimination, and identification scores averaged for both nasal sides. Gustatory function was assessed using the three-drop test resulting in the gustatory composite score (CSg). The data were compared with SOF and SGF. RESULTS: Of 43 adult convalescents (median age: 68 years; 58% female) after SARS-CoV­2 infection, 18 participants (42%) had olfactory complaints due to SOF, one participant (2%) complained of taste disturbance due to SGF. The TDIa was 22.0 ± 5.9. Normosmia, hyposmia, and anosmia were seen in 17, 18, and eight participants, respectively. TDIa correlated with SOF (rs = -0.434, p = 0.004); CSg was 23.5 ± 2.7. Normogeusia and hypogeusia were objectified in 39 and four participants, respectively. The prevalence of long-term olfactory dysfunction and gustatory dysfunction in the study group was 60.5 and 9.3%, respectively. CONCLUSION: The SOF was reliable, especially for participants who felt a sudden chemosensory dysfunction during the outbreak. At 4 months after SARS-CoV­2 infection, a high proportion of participants were dysosmic, whereas nearly all of them had normal taste function.

Acute epileptiform activity induced by gabazine involves proteasomal rather than lysosomal degradation of KCa2.2 channels.

Voltage-independent, Ca2+-activated K+ channels (KCa2.2, previously named SK2) are typically activated during a train of action potentials, and hence, are powerful regulators of cellular excitability by generating an afterhyperpolarizing potential (AHP) following prolonged excitation. In the acute in vitro epilepsy model induced in hippocampal brain slice preparations by exposure to the GABAA receptor blocker gabazine (GZ), the AHP was previously shown to be significantly decreased. Here, we asked the question whether KCa2.2 protein degradation occurs in this model and which pathways are involved. To this end, we applied either gabazine alone or gabazine together with inhibitors of proteasomal and lysosomal protein degradation pathways, Z-Leu-Leu-Leu-CHO (MG132) and chloroquine (CQ), respectively. Western blot analysis showed a significant decrease of total KCa2.2 protein content in GZ-treated slices which could be rescued by concomitant incubation with MG132 and CQ. Using HEK293 cells transfected with a green fluorescent protein-tagged KCa2.2 construct, we demonstrated that proteasomal rather than lysosomal degradation was involved in KCa2.2 reduction. We then recorded epileptiform afterdischarges at hippocampal Schaffer collateral-CA1 synapses and confirmed that the GZ-induced increase was significantly attenuated by both MG132 and CQ, with MG132 being significantly more effective than CQ. Epileptiform afterdischarges were almost prevented by co-application of protein degradation inhibitors. Furthermore, epileptiform afterdischarges could be re-established by using the KCa2.2 blocker UCL 1684 suggesting involvement of KCa2.2. We conclude that in GZ-induced acute epilepsy, KCa2.2 degradation by proteasomal rather than lysosomal pathways plays a major role in the generation of epileptiform afterdischarges.

Functional Metaplasticity of Hippocampal Schaffer Collateral-CA1 Synapses Is Reversed in Chronically Epileptic Rats.

Spatial learning and associating spatial information with individual experience are crucial for rodents and higher mammals. Hence, studying the cellular and molecular cascades involved in the key mechanism of information storage in the brain, synaptic plasticity, has led to enormous knowledge in this field. A major open question applies to the interdependence between synaptic plasticity and its behavioral correlates. In this context, it has become clear that behavioral aspects may impact subsequent synaptic plasticity, a phenomenon termed behavioral metaplasticity. Here, we trained control and pilocarpine-treated chronically epileptic rats of two different age groups (adolescent and adult) in a spatial memory task and subsequently tested long-term potentiation (LTP) in vitro at Schaffer collateral-CA1 synapses. As expected, memory acquisition in the behavioral task was significantly impaired both in pilocarpine-treated animals and in adult controls. Accordingly, these groups, without being tested in the behavioral training task, showed reduced CA1-LTP levels compared to untrained young controls. Spatial memory training significantly reduced subsequent CA1-LTP in vitro in the adolescent control group yet enhanced CA1-LTP in the adult pilocarpine-treated group. Such training in the adolescent pilocarpine-treated and adult control groups resulted in intermediate changes. Our study demonstrates age-dependent functional metaplasticity following a spatial memory training task and its reversal under pathological conditions.

Interplay between interictal spikes and behavioral seizures in young, but not aged pilocarpine-treated epileptic rats.

Interictal spike activity is commonly observed in the EEG of patients with epilepsy, but the causal interrelationship between interictal spikes and behavioral seizures is poorly understood. We performed long-term video-EEG monitoring of 16 epileptic rats after pilocarpine-induced status epilepticus and five control animals. To quantify the interplay between periods of spikes and seizures, we calculated the time spent with spikes as well as the time spent with seizures for each animal. Within a given subject, we found a significant correlation between these two measures in 7/11 young epileptic rats (<400 days); this correlation was positive in six cases and negative in one. By contrast, none of five aged pilocarpine-treated animals exhibited significant correlation coefficients between spike periods and seizures (>600 days, P<0.05). Instead, aged epileptic rats showed a prominent predominance for either spike periods or seizures, which might explain the absence of significant correlation in this population. We found that there is a significant interplay between interictal periods of spikes and behavioral seizures in young epileptic animals, but this association is absent during aging.

P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

AIM: Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. METHODS: Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. RESULTS: Application of the non-hydrolyzable P2 receptor agonists α,ß-Me-ATP or 2-Me-S-ADP (10, 100 µmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 µmol/L), PLC inhibitor U73122 (100 µmol/L), IP3 receptor blocker 2-APB (100 µmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 µmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,ß-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 µmol/L) or P2Y13 receptor antagonist MRS2211 (100 µmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 µmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 µmol/L) not only abolished α,ß-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. CONCLUSION: P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

Status Epilepticus Enhances Depotentiation after Fully Established LTP in an NMDAR-Dependent but GluN2B-Independent Manner.

N-Methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) can be reversed by low-frequency stimulation (LFS) referred to as depotentiation (DP). We previously found GluN2B upregulated in CA1 neurons from post-status epilepticus (post-SE) tissue associated with an enhanced LTP. Here, we tested whether LFS-induced DP is also altered in pathological GluN2B upregulation. Although LTP was enhanced in post-SE tissue, LTP was significantly reversed in this tissue, but not in controls. We next tested the effect of the GluN2B subunit-specific blocker Ro 25-6981 (1 µM) on LFS-DP. As expected, LFS had no effect on synaptic strength in the presence of the GluN2B blocker in control tissue. In marked contrast, LFS-DP was also attained in post-SE tissue indicating that GluN2B was obviously not involved in depotentiation. To test for NMDA receptor-dependence, we applied the NMDA receptor antagonist D-AP5 (50 µM) prior to LFS and observed that DP was abolished in both control and post-SE tissue confirming NMDA receptor involvement. These results indicate that control Schaffer collateral-CA1 synapses cannot be depotentiated after fully established LTP, but LFS was able to reverse LTP significantly in post-SE tissue. However, while LFS-DP clearly required NMDA receptor activation, GluN2B-containing NMDA receptors were not involved in this form of depotentiation.

Inverse relationship of Rho kinase and myosin-light chain kinase expression in the aging human detrusor smooth muscle.

BACKGROUND: Rho kinase (ROCK) and myosin-light chain kinase (MLCK) are key enzymes in smooth muscle contraction. Previous data have suggested that ROCK contribution to human detrusor contraction is increasing with age. Here, we have analyzed the transcriptional expression of Rho kinase isoforms (ROCK1 and ROCK2) as well as MLCK in the aging human detrusor smooth muscle obtained from resected tissue. METHODS: Small pieces of macroscopically healthy human detrusor smooth muscle (urothelium-free) were prepared for quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Transcript expression (mRNA level) of the target genes ROCK1, ROCK2 and MLCK was normalized to three common reference genes (glyceraldehyde-3-phosphate dehydrogenase, ß-actin, phosphoglycerate kinase 1). RESULTS: We found that across all ages the expression level of ROCK (i.e. ROCK1 and ROCK2 together) was almost equal to that of MLCK in the human bladder. Further, ROCK2 showed a significantly higher expression level than ROCK1. Among all subjects, there was no significant correlation of any single target gene to age, but expression levels of ROCK and MLCK were inversely correlated. Moreover, the within-subject analysis revealed that the ROCK-to-MLCK ratio showed a significantly negative correlation to age. Thus, within a given subject, there is a relative ROCK down-regulation and concomitant MLCK up-regulation. CONCLUSIONS: Together with previous data in human detrusor specimens showing increased ROCK contribution to detrusor contraction, we speculate that the drop of the ROCK-to-MLCK ratio may occur as an attempt to compensate for the increased Rho kinase activity.

Age-related decrease of adenosine-mediated relaxation in rat detrusor is a result of A2B receptor downregulation.

OBJECTIVES: To analyze the effect of adenosine on detrusor smooth muscle contraction and to assess age-related changes of adenosine function. METHODS: Sustained contractions were induced in young (10-30 days) and old (>60 days) rat detrusor muscle strips by application of 30 mmol/L K(+) and adenosine (0.1-400 µmol/L), which was either applied before raising the K(+) concentration or added to the precontracted muscle strip. Quantitative polymerase chain reaction analyses were used to study adenosine receptor expression in rat and human detrusor specimens. RESULTS: Pretreatment with adenosine dose-dependently reduced subsequent K(+) -induced contraction in detrusor muscle strips from young rats (half-maximal effect = 40 µmol/L). The residual depolarization-induced contraction strength in young tissue was significantly smaller than in tissue from old animals, showing a greater potency of adenosine in young detrusor samples. Likewise, the relaxing effect of adenosine on precontracted detrusor muscle was also significantly more pronounced in young compared with older detrusor. Quantitative polymerase chain reaction showed an age-related downregulation of the adenosine A2B receptor in rat detrusor tissues, which could be confirmed in human detrusor samples. Furthermore, relaxation of both K(+) -induced as well as carbachol-induced contraction by the specific A2B receptor agonist BAY 60-6583 was significantly more pronounced in young than in old rats. CONCLUSIONS: Adenosine powerfully counteracts contraction of detrusor smooth muscle, which is lost in the aging bladder. This is paralleled by an age-dependent transcriptional downregulation of the low-affinity A2B receptor. Hence, this might be pathophysiologically relevant in conditions of raised adenosine concentrations, such as hyperactive bladder contractility.

Impaired D-serine-mediated cotransmission mediates cognitive dysfunction in epilepsy.

The modulation of synaptic plasticity by NMDA receptor (NMDAR)-mediated processes is essential for many forms of learning and memory. Activation of NMDARs by glutamate requires the binding of a coagonist to a regulatory site of the receptor. In many forebrain regions, this coagonist is d-serine. Here, we show that experimental epilepsy in rats is associated with a reduction in the CNS levels of d-serine, which leads to a desaturation of the coagonist binding site of synaptic and extrasynaptic NMDARs. In addition, the subunit composition of synaptic NMDARs changes in chronic epilepsy. The desaturation of NMDARs causes a deficit in hippocampal long-term potentiation, which can be rescued with exogenously supplied d-serine. Importantly, exogenous d-serine improves spatial learning in epileptic animals. These results strongly suggest that d-serine deficiency is important in the amnestic symptoms of temporal lobe epilepsy. Our results point to a possible clinical utility of d-serine to alleviate these disease manifestations.

In vivo treatment with the casein kinase 2 inhibitor 4,5,6,7- tetrabromotriazole augments the slow afterhyperpolarizing potential and prevents acute epileptiform activity.

OBJECTIVE: The slow afterhyperpolarizing potential (sAHP) following prolonged depolarization is a major intrinsic mechanism of neuronal inhibition, by powerfully dampening excitability for up to 2 s. Therefore, an altered sAHP function might be vulnerable to hyperexcitable states such as epilepsy. Here, we have investigated the role of casein kinase 2 (CK2) on the sAHP in control and chronically epileptic tissue. METHODS: Using the rat pilocarpine model of chronic temporal lobe epilepsy, we performed whole-cell patch-clamp recordings of acutely isolated CA1 pyramidal cells and field potential measurements on hippocampal slices. RESULTS: Chronic oral administration of the CK2 inhibitor 4,5,6,7-tetrabromotriazole (TBB) for 4 days prior to brain dissection caused a significant increase of the sAHP-mediating current in both control and epileptic tissues. In contrast, when TBB was acutely applied during the patch-clamp recording, the sAHP remained unaltered, indicating that chronic CK2 inhibition was required for sAHP augmentation. To test whether CK2 inhibition also has an anticonvulsive effect, we evoked recurrent epileptiform discharges (REDs) in hippocampal slice preparations by Mg²âº removal. It is important to note that chronic oral TBB administration abolished REDs induced by 0-Mg²âº solution, suggesting that CK2 inhibition indeed has anticonvulsive and perhaps antiepileptogenic properties. SIGNIFICANCE: Our data demonstrated that CK2 inhibition augments the sAHP and might represent a novel mechanism of action of anticonvulsant drugs.

Age-dependent contribution of Rho kinase in carbachol-induced contraction of human detrusor smooth muscle in vitro.

AIM: Activation of muscarinic receptors on the detrusor smooth muscle is followed by contraction, which involves both myosin light chain kinase (MLCK) and Rho kinase (ROCK). The aim of this study was to determine the relative contributions of MLCK and ROCK to carbachol-induced contraction of human detrusor smooth muscle in vitro. METHODS: Detrusor smooth muscle strips were prepared from the macroscopically unaffected bladder wall of patients underwent cystectomy. The strips were fixed in an organ bath, and carbachol or KCl-induced isometric contractions were measured by force transducers. RESULTS: Addition of carbachol (0.4-4 µmol/L) into the bath induced concentration-dependent contractions of detrusor specimens, which was completely abolished by atropine (1 µmol/L). Pre-incubation of detrusor specimens with either the MLCK inhibitor ML-9 or the ROCK inhibitors HA1100 and Y-27632 (each at 10 µmol/L) significantly blocked carbachol-induced contractions as compared to the time-control experiments. Moreover, MLCK and ROCK inhibition were equally effective in reducing carbachol-induced contractions. The residual carbachol-induced contractions in the presence of both MLCK and ROCK inhibitors were significantly smaller than the contractions obtained when only one enzyme (either MLCK or ROCK) was inhibited, suggesting an additive effect of the two kinases. Interestingly, ROCK-mediated carbachol-induced contractions were positively correlated to the age of patients (r=o.52, P<0.05). CONCLUSION: Both MLCK and ROCK contribute to carbachol-induced contractions of human detrusor smooth muscle. ROCK inhibitors may be a new pharmacological approach to modulate human bladder hyperactivity.

NMDA receptor-dependent metaplasticity by high-frequency magnetic stimulation.

High-frequency magnetic stimulation (HFMS) can elicit N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) at Schaffer collateral-CA1 pyramidal cell synapses. Here, we investigated the priming effect of HFMS on the subsequent magnitude of electrically induced LTP in the CA1 region of rat hippocampal slices using field excitatory postsynaptic potential (fEPSP) recordings. In control slices, electrical high-frequency conditioning stimulation (CS) could reliably induce LTP. In contrast, the same CS protocol resulted in long-term depression when HFMS was delivered to the slice 30 min prior to the electrical stimulation. HFMS-priming was diminished when applied in the presence of the metabotropic glutamate receptor antagonists (RS)-α-methylserine-O-phosphate (MSOP) and (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Moreover, when HFMS was delivered in the presence of the NMDA receptor-antagonist D-2-amino-5-phosphonovalerate (50 µM), CS-induced electrical LTP was again as high as under control conditions in slices without priming. These results demonstrate that HFMS significantly reduced the propensity of subsequent electrical LTP and show that both metabotropic glutamate and NMDA receptor activation were involved in this form of HFMS-induced metaplasticity.

Inhibition of Acute mGluR5-Dependent Depression in Hippocampal CA1 by High-Frequency Magnetic Stimulation.

High-frequency magnetic stimulation (HFMS) applied directly to the hippocampal slice preparation in vitro induces activity-dependent synaptic plasticity and metaplasticity. In addition, changes in synaptic transmission following HFMS involve the activation of N-methyl-D-aspartate and metabotropic glutamate receptors (mGluR). Here, we asked whether a short period of HFMS (5 × 10 delta-burst trains, duration of ~1 min) could alter mGluR5-mediated depression at Schaffer collateral-CA1 synapses in the acute brain slice preparation at 30 min after HFMS. To this end, we obtained field excitatory postsynaptic potential (fEPSP) slopes from Schaffer collateral-CA1 synapses after HFMS or control. First, we demonstrated that activity-dependent plasticity following HFMS depends on the slice orientation towards the magnetic coil indicating specific ion fluxes induced by magnetic fields. Second, we found that the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine reduced the field excitatory postsynaptic potential (fEPSP) slopes in control slices but rather enhanced them in HFMS-treated slices. In contrast, the compound (S)-3,5-dihydroxyphenylglycine acting at both mGluR1 and mGluR5 reduced fEPSP slopes in both control and HFMS-treated slices. Importantly, the mGluR-dependent effects were independent from the slice-to-coil orientation indicating that asynchronous glutamate release could play a role. We conclude that a short period of HFMS inhibits subsequently evoked mGluR5-dependent depression at Schaffer collateral-CA1 synapses. This could be relevant for repetitive transcranial magnetic stimulation in psychiatric disorders such as major depression.

Persistent Kv7.2/7.3 downregulation in the rat pilocarpine model of mesial temporal lobe epilepsy.

Mutations within the Kv7.2 and Kv7.3 genes are well described causes for genetic childhood epilepsies. Knowledge on these channels in acquired focal epilepsy, especially in mesial temporal lobe epilepsy (mTLE), however, is scarce. Here, we used the rat pilocarpine model of drug-resistant mTLE to elucidate both expression and function by quantitative polymerase-chain reaction, immunohistochemistry, and electrophysiology, respectively. We found transcriptional downregulation of Kv7.2 and Kv7.3 as well as reduced Kv7.2 expression in epileptic CA1. Consequences were altered synaptic transmission, hyperexcitability which consisted of epileptiform afterpotentials, and increased susceptibility to acute GABAergic disinhibition. Importantly, blocking Kv7 channels with XE991 increased hyperexcitability in control tissue, but not in chronically epileptic tissue suggesting that the Kv7 deficit had precluded XE991 effects in this tissue. Conversely, XE991 resulted in comparable reduction of the paired-pulse ratio in both experimental groups implying preserved presynaptic Kv7.2 function of Schaffer collateral terminals. Consistent with Kv7.2/7.3 downregulation, the Kv7.3 channel opener ß-hydroxybutyrate failed to mitigate hyperexcitability. Our findings demonstrate that compromised Kv7 function is not only relevant in genetic epilepsy, but also in acquired focal epilepsy. Moreover, they help explain reduced anti-seizure efficacy of Kv7 channel openers in drug-resistant epilepsy.

A glutamatergic biomarker panel enables differentiating Grade 4 gliomas/astrocytomas from brain metastases.

Background: The differentiation of high-grade glioma and brain tumors of an extracranial origin is eminent for the decision on subsequent treatment regimens. While in high-grade glioma, a surgical resection of the tumor mass is a fundamental part of current standard regimens, in brain metastasis, the burden of the primary tumor must be considered. However, without a cancer history, the differentiation remains challenging in the imaging. Hence, biopsies are common that may help to identify the tumor origin. An additional tool to support the differentiation may be of great help. For this purpose, we aimed to identify a biomarker panel based on the expression analysis of a small sample of tissue to support the pathological analysis of surgery resection specimens. Given that an aberrant glutamate signaling was identified to drive glioblastoma progression, we focused on glutamate receptors and key players of glutamate homeostasis. Methods: Based on surgically resected samples from 55 brain tumors, the expression of ionotropic and metabotropic glutamate receptors and key players of glutamate homeostasis were analyzed by RT-PCR. Subsequently, a receiver operating characteristic (ROC) analysis was performed to identify genes whose expression levels may be associated with either glioblastoma or brain metastasis. Results: Out of a total of 29 glutamatergic genes analyzed, nine genes presented a significantly different expression level between high-grade gliomas and brain metastases. Of those, seven were identified as potential biomarker candidates including genes encoding for AMPA receptors GRIA1, GRIA2, kainate receptors GRIK1 and GRIK4, metabotropic receptor GRM3, transaminase BCAT1 and the glutamine synthetase (encoded by GLUL). Overall, the biomarker panel achieved an accuracy of 88% (95% CI: 87.1, 90.8) in predicting the tumor entity. Gene expression data, however, could not discriminate between patients with seizures from those without. Conclusion: We have identified a panel of seven genes whose expression may serve as a biomarker panel to discriminate glioblastomas and brain metastases at the molecular level. After further validation, our biomarker signatures could be of great use in the decision making on subsequent treatment regimens after diagnosis.

Enhanced NMDA receptor-dependent LTP in the epileptic CA1 area via upregulation of NR2B.

Impairment of synaptic plasticity such as long-term potentiation (LTP) is a common finding in various animal models of a number of neurodegenerative disorders. While cognitive deficits associated with these models are plausibly attributed to impaired plasticity, it is an intriguing question whether learning impairment correlates in general with compromised synaptic plasticity. In the present study, we have addressed this issue and discovered an enhancement of theta-burst stimulation-induced LTP at Schaffer collateral-CA1 synapses from chronically epileptic animals. The LTP enhancement was abolished by the NMDA receptor 2B (NR2B) blocker Ro 25-6981 (1µM) while it was preserved following application of the NR2A blocker NVP-AAM077 (50nM). Moreover, pharmacological characterization of intracellularly recorded excitatory postsynaptic potentials (EPSP) from CA1 pyramidal neurons indicated an increased NR2B/NR2A ratio in epileptic tissue, and NMDA receptor mediated excitatory postsynaptic currents showed significantly longer decay times. Quantitative reverse-transcriptase PCR confirmed the transcriptional up-regulation of NR2B-mRNA in chronically epileptic animals. To test the significance for epileptiform activity, recurrent epileptiform discharges (REDs) in the CA1 area induced by bath application of either high K(+) (8mM) plus gabazine (5µM) or 4-aminopyridine (50µM), were also characterized pharmacologically. While in control slices the presence of Ro 25-6981 had no effect on the RED frequency, NR2B inhibition significantly increased epileptic activity in tissue from epileptic animals. Our results demonstrate that CA1 synapses in chronically epileptic tissue can undergo an LTP enhancement due to an NR2B up-regulation in CA1 pyramidal neurons. On the network level, this up-regulation appears to be a compensatory process, since blockade of these receptors leaves the tissue more susceptible to hyperexcitability.