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.

Effects of Ginkgo biloba Extract EGb 761, Donepezil and their Combination on Central Cholinergic Function in Aged Rats.

PURPOSE: Ginkgo extract EGb 761 and cholinesterase inhibitors have been shown to be effective in the treatment of dementia patients. In addition to neuroprotective effects, Ginkgo extract EGb 761 has been reported to elevate brain levels of certain neurotransmitters such as dopamine, noradrenaline, and acetylcholine. In the present study, we investigated the impact of EGb 761, donepezil and the combination of both drugs on the central cholinergic system in aged rats. METHODS: 24 month old rats received EGb 761 (100 mg/kg/day), donepezil (1.5 mg/kg/day), the combination of both drugs or vehicle control by oral gavage for 14 days. We used microdialysis in rat hippocampus to monitor extracellular concentrations of acetylcholine (ACh), choline, glucose and lactate. Brain homogenates were prepared to measure activities of acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and high affinity choline uptake (HACU). RESULTS: While EGb 761 alone had no effect, donepezil and the combination of donepezil and EGb 761 increased basal ACh levels by 2- to 3-fold. Concomitantly, significant reductions of AChE and HACU were measured in both groups. No differences were seen between donepezil and the combination in these parameters. Treatment with EGb 761 decreased extracellular choline release and showed a tendency to moderately elevate ChAT activity. CONCLUSIONS: We found that donepezil and EGb 761 do not display a pharmacological interaction when given together. Adding EGb 761 did not modify the effects of donepezil on the hippocampal cholinergic system. Reduced choline levels indicate neuroprotective properties of EGb 761. Therefore, the combination of EGb 761 and donepezil may be beneficial in the treatment of Alzheimer's disease (AD). This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Dynamics of hippocampal acetylcholine release during lithium-pilocarpine-induced status epilepticus in rats.

The lithium-pilocarpine model is a rat model of epilepsy that mimics status epilepticus in humans. Here, we report changes of acetylcholine (ACh) release in the hippocampus before, during and after status epilepticus as monitored by microdialysis in unanesthetized rats. Administration of pilocarpine (30 mg/kg s.c.) to rats pretreated with lithium chloride (127 mg/kg i.p.) caused a massive, six-fold increase of hippocampal ACh release, paralleling the development of tonic seizures. When seizures were stopped by administration of diazepam (10 mg/kg i.p.) or ketamine (75 mg/kg i.p.), ACh levels returned to normal. Extracellular concentrations of glutamate remained unchanged during this procedure. Administration of atropine (1 mg/kg i.p.) 2 h after pilocarpine caused a further increase of ACh but did not affect seizures, whereas injection of mecamylamine (5 mg/kg i.p.) reduced ACh levels and seizures in a delayed fashion. Local infusion of tetrodotoxin, 1 µM locally) or hemicholinium (10 µM locally) strongly reduced ACh release and had delayed effects on seizures. Administration of glucose or inositol (250 mg/kg each i.p.) had no visible consequences. In parallel experiments, lithium-pilocarpine-induced status epilepticus also enhanced striatal ACh release, and hippocampal ACh levels equally increased when status epilepticus was induced by kainate (30 mg/kg i.p.). Taken together, our results demonstrate that seizure development in status epilepticus models is accompanied by massive increases of extracellular ACh, but not glutamate, levels. Treatments that reduce seizure activity also reliably reduce extracellular ACh levels.

Acetylcholine and choline in honey bee (Apis mellifera) worker brood food are seasonal and age-dependent.

Nursing honeybees produce brood food with millimolar concentrations of acetylcholine (ACh), which is synthesized through head gland secretions mixed with honey stomach contents. While we previously demonstrated the necessity of ACh for proper larval development, the dynamics of ACh levels throughout ontogenesis and their seasonal variations have remained unclear until now. Our HPLC analysis reveals dependencies of choline and ACh levels on larval development days (LDDs), influenced by seasonal (April-September) variations. Median ACh concentrations peak on LDD 2, declining significantly toward cell capping, while choline levels are lowest during the initial LDDs, rising markedly toward cell capping. Seasonal patterns show peak ACh levels from April to June and a low in August, paralleling choline's peak in July and low in August. This seasonality holds consistently across multiple years (2020-2022) and colonies, despite potential variations in colony performance and environmental conditions. Our analysis found no correlation between temperature, sunshine, precipitation, or favourable foraging days and ACh/choline levels, suggesting the involvement of additional factors. These findings underscore the seasonal fluctuation of ACh levels and its potential implications for the genetic programs governing winter bee development.

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.

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.

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.

Staurosporine-induced apoptosis presents with unexpected cholinergic effects in a differentiated neuroblastoma cell line.

Apoptosis of cholinergic neurons is one of the core hallmarks of Alzheimer's disease. SH-SY5Y neuroblastoma cells differentiated to the cholinergic phenotype were exposed to 100nM staurosporine. Over a treatment period of 24h, the pro- and anti-apoptotic factors, caspase-3 and Bcl-2, as well as LDH release as a measure of cell viability, were assessed in conjunction with the number of apoptotic cells by means of fluorescence-activated cell sorting. Caspase-3 activity and LDH release increased by 30% and 20% over controls, respectively, while Bcl-2 levels rose by 200% over controls. Furthermore, staurosporine treatment resulted in decreased acetylcholinesterase (AChE) enzymatic activity and decreased protein levels of the AChE splice variant tailed AChE (AChE-T). Only a slight increase in levels of readthrough AChE (AChE-R) was observed. Likewise, staurosporine reduced levels and activity of the cholinergic players choline acetyltransferase and high affinity choline uptake. The present study demonstrates that treatment with staurosporine leads to apoptotic events, which, however, are not reflected in the increased AChE activity and the alterations of AChE isoforms expression that are usually seen in apoptotic conditions. The effects of various additional phosphorylation inhibitors on AChE activity suggest that these unexpected cholinergic effects, firstly, are linked to the impact of staurosporine on phosphorylation and, secondly, reveal themselves in a first phase of cellular adaption that precedes neurotoxicity and subsequent cell death.