Oxidative stress in rat brain during experimental status epilepticus: effect of antioxidants.

Antioxidants have been proposed as a treatment for diseases of the central nervous system. However, few studies actually studied their effects in the brain. To test central actions of antioxidants, we used the lithium-pilocarpine (Li-Pilo) model of status epilepticus (SE) in the rat in which seizures are accompanied by significant oxidative stress. We used in vivo microdialysis to determine isoprostane levels during SE in real time and brain homogenates for other measures of oxidative stress. Six different antioxidants were tested in acute and preventive experiments (vitamin C, vitamin E, ebselen, resveratrol, n-tert-butyl-α-phenylnitrone and coenzyme Q10). None of the antioxidants had an effect when given acutely during SE. In contrast, when antioxidants were given for 3 days prior to seizure induction, vitamins C and E reduced isoprostane formation by 58% and 65%, respectively. Pretreatment with the other antioxidants was ineffective. In brain homogenates prepared after 90 min of seizures, SE decreased the ratio of reduced vs. oxidized glutathione (GSH/GSSG ratio) from 60.8 to 7.50 and caused a twofold increase of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and protein carbonyls. Pretreatment with vitamin C or vitamin E mitigated these effects and increased the GSH/GSSG ratio to 23.9 and 28.3, respectively. Again, the other antioxidants were not effective. We conclude that preventive treatment with vitamin C or vitamin E ameliorates seizure-induced oxidative damage in the brain. Several well-studied antioxidants were inactive, possibly due to limited brain permeability or a lack of chain-breaking antioxidant activity in hydrophilic compounds.

Mitochondrial Effects of Hydromethylthionine, Rivastigmine and Memantine in Tau-Transgenic Mice.

Tau protein aggregations are important contributors to the etiology of Alzheimer's disease (AD). Hydromethylthionine (HMT) is a potent inhibitor of tau aggregation in vitro and in vivo and is being developed as a possible anti-dementia medication. HMT was also shown to affect the cholinergic system and to interact with mitochondria. Here, we used tau-transgenic (L1 and L66) and wild-type NMRI mice that were treated with HMT, rivastigmine and memantine and with combinations thereof, for 2-4 weeks. We measured HMT concentrations in both brain homogenates and isolated mitochondria and concentrations of glucose, lactate and pyruvate in brain by microdialysis. In isolated brain mitochondria, we recorded oxygen consumption of mitochondrial complexes by respirometry. While rivastigmine and memantine lowered mitochondrial respiration, HMT did not affect respiration in wild-type animals and increased respiration in tau-transgenic L1 mice. Glucose and lactate levels were not affected by HMT administration. The presence of HMT in isolated mitochondria was established. In summary, traditional anti-dementia drugs impair mitochondrial function while HMT has no adverse effects on mitochondrial respiration in tau-transgenic mice. These results support the further development of HMT as an anti-dementia drug.

Inhibition of mitochondrial respiration by general anesthetic drugs.

General anesthetic drugs have been associated with various unwanted effects including an interference with mitochondrial function. We had previously observed increases of lactate formation in the mouse brain during anesthesia with volatile anesthetic agents. In the present work, we used mitochondria that were freshly isolated from mouse brain to test mitochondrial respiration and ATP synthesis in the presence of six common anesthetic drugs. The volatile anesthetics isoflurane, halothane, and (to a lesser extent) sevoflurane caused an inhibition of complex I of the electron transport chain in a dose-dependent manner. Significant effects were seen at concentrations that are reached under clinical conditions (< 0.5 mM). Pentobarbital and propofol also inhibited complex I but at concentrations that were two-fold higher than clinical EC50 values. Only propofol caused an inhibition of complex II. Complex IV respiration was not affected by either agent. Ketamine did not affect mitochondrial respiration. Similarly, all anesthetic agents except ketamine suppressed ATP production at high concentrations. Only halothane increased cytochrome c release indicating damage of the mitochondrial membrane. In summary, volatile general anesthetic agents as well as pentobarbital and propofol dose-dependently inhibit mitochondrial respiration. This action may contribute to depressive actions of the drugs in the brain.

ß-Hydroxybutyrate Improves Mitochondrial Function After Transient Ischemia in the Mouse.

ß-Hydroxybutyrate (BHB) is a ketone body formed in high amounts during lipolysis and fasting. Ketone bodies and the ketogenic diet were suggested as neuroprotective agents in neurodegenerative disease. In the present work, we induced transient ischemia in mouse brain by unilaterally occluding the middle cerebral artery for 90 min. BHB (30 mg/kg), given immediately after reperfusion, significantly improved the neurological score determined after 24 h. In isolated mitochondria from mouse brain, oxygen consumption by the complexes I, II and IV was reduced immediately after ischemia but recovered slowly over 1 week. The single acute BHB administration after reperfusion improved complex I and II activity after 24 h while no significant effects were seen at later time points. After 24 h, plasma and brain BHB concentrations were strongly increased while mitochondrial intermediates (citrate, succinate) were unchanged in brain tissue. Our data suggest that a single administration of BHB may improve mitochondrial respiration for 1-2 days but not for later time points. Endogenous BHB formation seems to complement the effects of exogenous BHB administration.

Cysteinyl leukotrienes and acetylcholine are biliary tuft cell cotransmitters.

The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.

Hydromethylthionine enhancement of central cholinergic signalling is blocked by rivastigmine and memantine.

The prevention of tau protein aggregations is a therapeutic goal for the treatment of Alzheimer's disease (AD), and hydromethylthionine (HMT) (also known as leucomethylthioninium-mesylate [LMTM]), is a potent inhibitor of tau aggregation in vitro and in vivo. In two Phase 3 clinical trials in AD, HMT had greater pharmacological activity on clinical endpoints in patients not receiving approved symptomatic treatments for AD (acetylcholinesterase (AChE) inhibitors and/or memantine) despite different mechanisms of action. To investigate this drug interaction in an animal model, we used tau-transgenic L1 and wild-type NMRI mice treated with rivastigmine or memantine prior to adding HMT, and measured changes in hippocampal acetylcholine (ACh) by microdialysis. HMT given alone doubled hippocampal ACh levels in both mouse lines and increased stimulated ACh release induced by exploration of the open field or by infusion of scopolamine. Rivastigmine increased ACh release in both mouse lines, whereas memantine was more active in tau-transgenic L1 mice. Importantly, our study revealed a negative interaction between HMT and symptomatic AD drugs: the HMT effect was completely eliminated in mice that had been pre-treated with either rivastigmine or memantine. Rivastigmine was found to inhibit AChE, whereas HMT and memantine had no effects on AChE or on choline acetyltransferase (ChAT). The interactions observed in this study demonstrate that HMT enhances cholinergic activity in mouse brain by a mechanism of action unrelated to AChE inhibition. Our findings establish that the drug interaction that was first observed clinically has a neuropharmacological basis and is not restricted to animals with tau aggregation pathology. Given the importance of the cholinergic system for memory function, the potential for commonly used AD drugs to interfere with the treatment effects of disease-modifying drugs needs to be taken into account in the design of clinical trials.

Effects of Magnesium Orotate, Benfotiamine and a Combination of Vitamins on Mitochondrial and Cholinergic Function in the TgF344-AD Rat Model of Alzheimer's Disease.

Glucose hypometabolism, mitochondrial dysfunction, and cholinergic deficits have been reported in early stages of Alzheimer's disease (AD). Here, we examine these parameters in TgF344-AD rats, an Alzheimer model that carries amyloid precursor protein and presenilin-1 mutations, and of wild type F344 rats. In mitochondria isolated from rat hippocampi, we found reductions of complex I and oxidative phosphorylation in transgenic rats. Further impairments, also of complex II, were observed in aged (wild-type and transgenic) rats. Treatment with a "cocktail" containing magnesium orotate, benfotiamine, folic acid, cyanocobalamin, and cholecalciferol did not affect mitochondrial activities in wild-type rats but restored diminished activities in transgenic rats to wild-type levels. Glucose, lactate, and pyruvate levels were unchanged by age, genetic background, or treatment. Using microdialysis, we also investigated extracellular concentrations of acetylcholine that were strongly reduced in transgenic animals. Again, ACh levels in wild-type rats did not change upon treatment with nutrients, whereas the cocktail increased hippocampal acetylcholine levels under physiological stimulation. We conclude that TgF344-AD rats display a distinct mitochondrial and cholinergic dysfunction not unlike the findings in patients suffering from AD. This dysfunction can be partially corrected by the application of the "cocktail" which is particularly active in aged rats. We suggest that the TgF344-AD rat is a promising model to further investigate mitochondrial and cholinergic dysfunction and potential treatment approaches for AD.

Fluorovinylsulfones and -Sulfonates as Potent Covalent Reversible Inhibitors of the Trypanosomal Cysteine Protease Rhodesain: Structure-Activity Relationship, Inhibition Mechanism, Metabolism, and In Vivo Studies.

Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, α-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (Ki = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo[b][1,4]dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC50 values (0.14/0.80 µM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations.

New Pathways for the Skin's Stress Response: The Cholinergic Neuropeptide SLURP-1 Can Activate Mast Cells and Alter Cytokine Production in Mice.

Background: The alpha7 nicotinic acetylcholine receptor (Chrna7) plays an essential anti-inflammatory role in immune homeostasis and was recently found on mast cells (MC). Psychosocial stress can trigger MC hyperactivation and increases pro-inflammatory cytokines in target tissues such as the skin. If the cholinergic system (CS) and Chrna7 ligands play a role in these cascades is largely unknown. Objective: To elucidate the role of the CS in the response to psychosocial stress using a mouse-model for stress-triggered cutaneous inflammatory circuits. Methods: Key CS markers (ACh, Ch, SLURP-1, SLURP-2, Lynx1, Chrm3, Chrna7, Chrna9, ChAT, VAChT, Oct3, AChE, and BChE) in skin and its MC (sMC), MC activation, immune parameters (TNFα, IL1ß, IL10, TGFß, HIF1α, and STAT3) and oxidative stress were analyzed in skin from 24 h noise-stressed mice and in cultured MC (cMC) from C57BL/6 or Chrna7-Knockout mice. Results: First, Chrna7 and SLURP-1 mRNA were exclusively upregulated in stressed skin. Second, histomorphometry located Chrna7 and SLURP-1 in nerves and sMC and demonstrated upregulated contacts and increased Chrna7+ sMC in stressed skin, while 5 ng/mL SLURP-1 degranulated cMC. Third, IL1ß+ sMC were high in stressed skin, and while SLURP-1 alone had no significant effect on cMC cytokines, it upregulated IL1ß in cMC from Chrna7-KO and in IL1ß-treated wildtype cMC. In addition, HIF1α+ sMC were high in stressed skin and Chrna7-agonist AR-R 17779 induced ROS in cMC while SLURP-1 upregulated TNFα and IL1ß in cMC when HIF1α was blocked. Conclusions: These data infer that the CS plays a role in the regulation of stress-sensitive inflammatory responses but may have a surprising pro-inflammatory effect in healthy skin, driving IL1ß expression if SLURP-1 is involved.

Microdialysis for chronic exertional compartment syndrome: a pilot study.

BACKGROUND: Diagnosing chronic exertional compartment syndrome (CECS) is still a challenge. An increase in intramuscular pressure during and following exercise is accepted as the diagnostic standard. However, neither the methods used nor the interpretation of the obtained results are sufficiently standardized. METHODS: In the present pilot study, the metabolic state of CECS patients was investigated using microdialysis. We hypothesized that there was no difference in intramuscular concentrations of glucose, lactate, glutamate, and glycerol before and after exercise (H10) or between patients suffering from CECS and healthy control subjects (H20). This study was designed as an explorative case-control study (level of evidence III). Twelve patients suffering from CECS of the lower leg and six matched asymptomatic control subjects underwent microdialysis in the anterior (n = 7) or deep posterior compartment (n = 11) of the leg. Following ultrasound-guided insertion of the microdialysis catheters, 10-minute fractions of the dialysates were collected first during rest and then following fatigue- or pain-induced discontinuation of exercise. Dialysates were analysed for lactate, glucose, glutamate, and glycerol concentrations 6 × 10 min before and 6 × 10 min after exercise. RESULTS: Exercise-induced increases in lactate, glutamate, and glycerol concentrations were detected in both CECS patients and control subjects (all p < 0.001). No differences between CECS patients and control subjects were found by comparing the intramuscular glucose, lactate, glutamate, and glycerol concentrations at rest and following exercise (all p > 0.05). CONCLUSIONS: We found exercise-induced increases in the lactate, glutamate, and glycerol levels in skeletal muscle. However, the metabolic changes did not differentiate CECS patients from healthy subjects. TRIAL REGISTRATION: The registration trial number is DRKS00021589 on DRKS. 'Retrospectively registered'. Date of registration: April 4, 2020.

Cholinergic and metabolic effects of metformin in mouse brain.

Metformin is widely used as a first-line treatment for type 2 diabetes, but central effects of metformin have received little attention. When metformin (200 mg/kg i.p.) was administered to C57Bl6 mice, metformin concentration in cerebrospinal fluid peaked at 29 µM after 30 min but dropped quickly and was low at 90 min. In mouse hypothalamus sampled by microdialysis, systemically administered metformin caused minor and transient increases of acetylcholine, glucose and lactate while choline levels decreased. When metformin (0.2-10 mM) was locally infused via retrodialysis, there was a short-lasting increase of acetylcholine in the hypothalamus. Extracellular lactate levels in hypothalamus showed a massive increase upon metformin infusion while glucose levels decreased. In isolated mitochondria of mouse brain, metformin inhibited oxygen consumption and the activity of complex I. Inhibition of mitochondrial respiration likely explains lactate formation in the brain during metformin infusion which may cause lactic acidosis during metformin intoxication. The changes of cholinergic activity in the hypothalamus may be associated with appetite suppression observed during metformin treatment.

Central cholinergic function and metabolic changes in streptozotocin-induced rat brain injury.

As glucose hypometabolism in the brain is an early sign of Alzheimer´s dementia (AD), the diabetogenic drug streptozotocin (STZ) has been used to induce Alzheimer-like pathology in rat brain by intracereboventricular injection (icv-STZ). However, many details of the pathological mechanism of STZ in this AD model remain unclear. Here, we report metabolic and cholinergic effects of icv-STZ using microdialysis in freely moving animals. We found that icv-STZ at a dose of 3 mg/kg (2 × 1.5 mg/kg) causes overt toxicity reflected in body weight loss. Three weeks after STZ administration, histological examination revealed a high number of glial fibrillary acidic protein reactive cells in the hippocampus, accompanied by Fluoro-Jade C-positive cells in the CA1 region. Glucose and lactate levels in microdialysates were unchanged, but mitochondrial respiration measured ex vivo was reduced by 9%-15%. High-affinity choline uptake, choline acetyltransferase, and acetylcholine esterase (AChE) activities in the hippocampus were reduced by 16%, 28%, and 30%, respectively. Importantly, extracellular acetylcholine (ACh) levels in the hippocampus were unchanged and responded to behavioral and pharmacological challenges. In comparison, extracellular ACh levels and cholinergic parameters in the striatum were unchanged or slightly increased. We conclude that the icv-STZ model poorly reflects central cholinergic dysfunction, an important characteristic of dementia. The icv-STZ model may be more aptly described as an animal model of hippocampal gliosis.

Establishing Human Male and Female Models of Cholinergic Neurons via Neurokine-Mediated Differentiation of LA-N-2 and LA-N-5 Neuroblastoma Cells.

Cholinergic neurons control numerous primate-specific and sexually dimorphic brain functions. Here, we present our differentiation protocol for the closely related human female and male neuroblastoma-originated cell lines LA-N-2 and LA-N-5. Pro-cholinergic differentiation (with upregulation of choline acetyltransferase) of both lines can be achieved using neurokines such as ciliary neurotrophic factor (CNTF). Comparative RNA sequencing and mass spectrometry analyses between those two cell lines, supported by experimental intervention, will deepen our understanding of cholinergic systems in human psychiatric and neurologic disease. For complete details on the use and execution of this protocol, please refer to Lobentanzer et al. (2019).

Transfer RNA fragments replace microRNA regulators of the cholinergic poststroke immune blockade.

Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced 2 d after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by qRT-PCR validated the top six up-regulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes up-regulated the top six stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using a single-stranded RNA mimic induced down-regulation of immune regulator Z-DNA binding protein 1. In summary, we identified a "changing of the guards" between small RNA types that may systemically affect homeostasis in poststroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein and RNA level.

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Mechanisms of Anticholinesterase Interference with Tau Aggregation Inhibitor Activity in a Tau-Transgenic Mouse Model.

BACKGROUND: Symptomatic treatments of Alzheimer's Disease (AD) with cholinesterase inhibitors and/or memantine are relatively ineffective and there is a need for new treatments targeting the underlying pathology of AD. In most of the failed disease-modifying trials, patients have been allowed to continue taking symptomatic treatments at stable doses, under the assumption that they do not impair efficacy. In recently completed Phase 3 trials testing the tau aggregation inhibitor leuco-methylthioninium bis (hydromethanesulfonate) (LMTM), we found significant differences in treatment response according to whether patients were taking LMTM either as monotherapy or as an add-on to symptomatic treatments. METHODS: We have examined the effect of either LMTM alone or chronic rivastigmine prior to LMTM treatment of tau transgenic mice expressing the short tau fragment that constitutes the tangle filaments of AD. We have measured acetylcholine levels, synaptosomal glutamate release, synaptic proteins, mitochondrial complex IV activity, tau pathology and Choline Acetyltransferase (ChAT) immunoreactivity. RESULTS: LMTM given alone increased hippocampal Acetylcholine (ACh) levels, glutamate release from synaptosomal preparations, synaptophysin levels in multiple brain regions and mitochondrial complex IV activity, reduced tau pathology, partially restored ChAT immunoreactivity in the basal forebrain and reversed deficits in spatial learning. Chronic pretreatment with rivastigmine was found to reduce or eliminate almost all these effects, apart from a reduction in tau aggregation pathology. LMTM effects on hippocampal ACh and synaptophysin levels were also reduced in wild-type mice. CONCLUSION: The interference with the pharmacological activity of LMTM by a cholinesterase inhibitor can be reproduced in a tau transgenic mouse model and, to a lesser extent, in wild-type mice. Long-term pretreatment with a symptomatic drug alters a broad range of brain responses to LMTM across different transmitter systems and cellular compartments at multiple levels of brain function. There is, therefore, no single locus for the negative interaction. Rather, the chronic neuronal activation induced by reducing cholinesterase function produces compensatory homeostatic downregulation in multiple neuronal systems. This reduces a broad range of treatment responses to LMTM associated with a reduction in tau aggregation pathology. Since the interference is dictated by homeostatic responses to prior symptomatic treatment, it is likely that there would be similar interference with other drugs tested as add-on to the existing symptomatic treatment, regardless of the intended therapeutic target or mode of action. The present findings outline key results that now provide a working model to explain interference by symptomatic treatment.

Integrative Transcriptomics Reveals Sexually Dimorphic Control of the Cholinergic/Neurokine Interface in Schizophrenia and Bipolar Disorder.

RNA sequencing analyses are often limited to identifying lowest p value transcripts, which does not address polygenic phenomena. To overcome this limitation, we developed an integrative approach that combines large-scale transcriptomic meta-analysis of patient brain tissues with single-cell sequencing data of CNS neurons, short RNA sequencing of human male- and female-originating cell lines, and connectomics of transcription factor and microRNA interactions with perturbed transcripts. We used this pipeline to analyze cortical transcripts of schizophrenia and bipolar disorder patients. Although these pathologies show massive transcriptional parallels, their clinically well-known sexual dimorphisms remain unexplained. Our method reveals the differences between afflicted men and women and identifies disease-affected pathways of cholinergic transmission and gp130-family neurokine controllers of immune function interlinked by microRNAs. This approach may open additional perspectives for seeking biomarkers and therapeutic targets in other transmitter systems and diseases.

Impaired hippocampal and thalamic acetylcholine release in P301L tau-transgenic mice.

We evaluated acetylcholine release by microdialysis in 10 month old control and JNPL3 mice which carry a mutant tau gene (P301 L). Three brain regions were compared: hippocampus and thalamus which receive cholinergic input from the basal forebrain, and the red nucleus which receives cholinergic projections from brain stem nuclei. Cognitive and motor functions of the mice were largely normal. In microdialysis experiments, we found significant reductions in basal ACh levels in hippocampus and thalamus, but not in the red nucleus. ACh release was impaired most strongly (by 50%) when a physiological stimulus was applied, i.e. exploration of a novel environment, whereas most mice responded adequately with an increase of ACh release upon infusion of scopolamine. A strong reduction of scopolamine-mediated ACh release was seen after amyloid Aß42 peptide was administered into the hippocampus of tau-transgenic mice. Choline acetyltransferase activities were unchanged in tau-transgenic mice but acetylcholinesterase activities were increased in thalamus. Lactate and choline levels were increased in tau-transgenic mice but high-affinity choline uptake was slightly reduced. Our data suggest that even mild to moderate tau pathology in JNPL3 mice is able to depress cholinergic transmission in brain regions that receive input from the basal forebrain via long projection neurons. This impairment may be reinforced by amyloid peptide formation.

Effects of Three Anti-Seizure Drugs on Cholinergic and Metabolic Activity in Experimental Status Epilepticus.

PURPOSE: Status epilepticus (SE) is characterized by recurrent seizure activity and can be drug- resistant. Knowledge of neuronal and metabolic activity of the brain during SE may be helpful to improve medical care. We here report the effects of three anti-seizure drugs on changes of acetylcholine energy metabolites and oxidative stress during SE. METHODS: We used the lithium-pilocarpine model in rats to induce SE and in vivo- microdialysis to monitor cholinergic and metabolic activity in the hippocampus. We measured extracellular concentrations of acetylcholine, glucose, lactate, pyruvate, glycerol and isoprostanes before and during SE, and after acute treatment with pregabalin, valproic acid, and levetiracteam. RESULTS: Upon onset of  SE, acetylcholine (ACh) release increased six- to eightfold. Glucose was increased only transiently by 30% but lactate levels rose four-fold, and extracellular concentrations of glycerol ten-fold. Isoprostanes are markers of oxidative stress and increased more than 20-fold. Two hours after pilocarpine adminstration, rats were treated with pregabalin (100 mg/kg), levetiracetam (200 mg/kg) or valproic acid (400 mg/kg) by i.p. injection. All three drugs stopped seizure activity in a delayed fashion, but at the doses indicated, only animals that received levetiracetam reached consciousness. All drugs reduced ACh release within 60-120 minutes. Lactate/pyruvate ratios, glycerol and isoprostanne levels were also reduced significantly after drug administration. CONCLUSIONS: Hippocampal ACh release closely follows seizure activity in SE and is attenuated when SE subsides. Pregabalin, valproic acid and levetiracetam all terminate seizures in the rat SE model and attenuate cholinergic and metabolic changes within two hours.

Commonalities and differences in extracellular levels of hippocampal acetylcholine and amino acid neurotransmitters during status epilepticus and subsequent epileptogenesis in two rat models of temporal lobe epilepsy.

Chemically or electrically induced status epilepticus (SE) in rodents is a commonly used method for induction of epilepsy. Structural and functional changes in the hippocampus play a pivotal role in epileptogenesis induced by SE. Although cholinergic mechanisms have long been thought to play an important role in the onset and propagation of epileptic seizures, not much is known about the potential role of acetylcholine (ACh) in ictogenesis and epileptogenesis in SE models of temporal lobe epilepsy. Here we used in vivo microdialysis to determine extracellular levels of ACh and, for comparison, several amino acid transmitters in the ventral hippocampus during SE, epileptogenesis, and the chronic epileptic state in two rat models of SE-induced epilepsy. SE was either induced by lithium-pilocarpine or by sustained electrical stimulation of the basolateral amygdala (BLA). ACh increased during SE in both models. Pretreatment with the muscarinic receptor antagonist scopolamine before BLA stimulation reduced SE severity and duration. In contrast to ACh, no consistent changes in amino acid levels were found during SE in the two models. During epileptogenesis and the chronic epileptic state, the only commonalities found in both models were a decrease in ACh in epileptic rats during the chronic epileptic state and a decrease in aspartate during epileptogenesis. The data demonstrate complex, model-dependent alterations in extracellular levels of ACh and amino acid neurotransmitters and only few commonalities. Thus, data originating from only one model of post-SE epilepsy should not be generalized but may have a limited translational value for understanding ictogenesis or epileptogenesis.