ABSTRACT
Increased neuronal expression of the Na-K-2Cl cotransporter NKCC1 has been implicated in the generation of seizures and epilepsy. However, conclusions from studies on the NKCC1-specific inhibitor, bumetanide, are equivocal, which is a consequence of the multiple potential cellular targets and poor brain penetration of this drug. Here, we used Nkcc1 knockout (KO) and wildtype (WT) littermate control mice to study the ictogenic and epileptogenic effects of intrahippocampal injection of kainate. Kainate (0.23 µg in 50 nl) induced limbic status epilepticus (SE) in both KO and WT mice with similar incidence, latency to SE onset, and SE duration, but the number of intermittent generalized convulsive seizures during SE was significantly higher in Nkcc1 KO mice, indicating increased SE severity. Following SE, spontaneous recurrent seizures (SRS) were recorded by continuous (24/7) video/EEG monitoring at 0-1, 4-5, and 12-13 weeks after kainate, using depth electrodes in the ipsilateral hippocampus. Latency to onset of electrographic SRS and the incidence of electrographic SRS were similar in WT and KO mice. However, the frequency of electrographic seizures was lower whereas the frequency of electroclinical seizures was higher in Nkcc1 KO mice, indicating a facilitated progression from electrographic to electroclinical seizures during chronic epilepsy, and a more severe epileptic phenotype, in the absence of NKCC1. The present findings suggest that NKCC1 is dispensable for the induction, progression and manifestation of epilepsy, and they do not support the widely held notion that inhibition of NKCC1 in the brain is a useful strategy for preventing or modifying epilepsy.
Subject(s)
Epilepsy, Temporal Lobe/metabolism , Solute Carrier Family 12, Member 2/metabolism , Animals , Convulsants/toxicity , Disease Models, Animal , Epilepsy, Temporal Lobe/chemically induced , Female , Kainic Acid/toxicity , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , PhenotypeABSTRACT
OBJECTIVES: The loop diuretic bumetanide has been proposed previously as an adjunct treatment for neonatal seizures because bumetanide is thought to potentiate the action of γ-aminobutyric acid (GABA)ergic drugs such as phenobarbital by preventing abnormal intracellular accumulation of chloride and the subsequent "GABA shift." However, a clinical trial in neonates failed to demonstrate such a synergistic effect of bumetanide, most likely because this drug only poorly penetrates into the brain. This prompted us to develop lipophilic prodrugs of bumetanide, such as the N,N-dimethylaminoethyl ester of bumetanide (DIMAEB), which rapidly enter the brain where they are hydrolyzed by esterases to the parent compound, as demonstrated previously by us in adult rodents. However, it is not known whether esterase activity in neonates is sufficient to hydrolyze ester prodrugs such as DIMAEB. METHODS: In the present study, we examined whether esterases in neonatal serum of healthy term infants are capable of hydrolyzing DIMAEB to bumetanide and whether this activity is different from the serum of adults. Furthermore, to extrapolate the findings to brain tissue, we performed experiments with brain tissue and serum of neonatal and adult rats. RESULTS: Serum from 1- to 2-day-old infants was capable of hydrolyzing DIMAEB to bumetanide at a rate similar to that of serum from adult individuals. Similarly, serum and brain tissue of neonatal rats rapidly hydrolyzed DIMAEB to bumetanide. SIGNIFICANCE: These data provide a prerequisite for further evaluating the potential of bumetanide prodrugs as add-on therapy to phenobarbital and other antiseizure drugs as a new strategy for improving pharmacotherapy of neonatal seizures.
Subject(s)
Brain/enzymology , Bumetanide/metabolism , Esterases , Esters/metabolism , Prodrugs/metabolism , Animals , Animals, Newborn , Brain/metabolism , Female , Humans , Infant, Newborn , Male , Rats , Serum/enzymology , Serum/metabolismABSTRACT
OBJECTIVE: Neonatal seizures are the most frequent type of neurological emergency in newborn infants, often being a consequence of prolonged perinatal asphyxia. Phenobarbital is currently the most widely used antiseizure drug for treatment of neonatal seizures, but fails to stop them in ~50% of cases. In a neonatal hypoxia-only model based on 11-day-old (P11) rats, the NKCC1 inhibitor bumetanide was reported to potentiate the antiseizure activity of phenobarbital, whereas it was ineffective in a human trial in neonates. The aim of this study was to evaluate the effect of clinically relevant doses of bumetanide as add-on to phenobarbital on neonatal seizures in a noninvasive model of birth asphyxia in P11 rats, designed for better translation to the human term neonate. METHODS: Intermittent asphyxia was induced for 30 minutes by exposing the rat pups to three 7 + 3-minute cycles of 9% and 5% O2 at constant 20% CO2 . Drug treatments were administered intraperitoneally either before or immediately after asphyxia. RESULTS: All untreated rat pups had seizures within 10 minutes after termination of asphyxia. Phenobarbital significantly blocked seizures when applied before asphyxia at 30 mg/kg but not 15 mg/kg. Administration of phenobarbital after asphyxia was ineffective, whereas midazolam (0.3 or 1 mg/kg) exerted significant antiseizure effects when administered before or after asphyxia. In general, focal seizures were more resistant to treatment than generalized convulsive seizures. Bumetanide (0.3 mg/kg) alone or in combination with phenobarbital (15 or 30 mg/kg) exerted no significant effect on seizure occurrence. SIGNIFICANCE: The data demonstrate that bumetanide does not increase the efficacy of phenobarbital in a model of birth asphyxia, which is consistent with the negative data of the recent human trial. The translational data obtained with the novel rat model of birth asphyxia indicate that it is a useful tool to evaluate novel treatments for neonatal seizures.
Subject(s)
Asphyxia Neonatorum/drug therapy , Bumetanide/therapeutic use , Disease Models, Animal , Midazolam/therapeutic use , Phenobarbital/therapeutic use , Seizures/drug therapy , Animals , Animals, Newborn , Anticonvulsants/therapeutic use , Asphyxia Neonatorum/complications , Asphyxia Neonatorum/physiopathology , Female , Hypnotics and Sedatives/therapeutic use , Male , Rats , Rats, Wistar , Seizures/etiology , Seizures/physiopathology , Sodium Potassium Chloride Symporter Inhibitors/therapeutic use , Treatment OutcomeABSTRACT
Parkinson's disease (PD) is a progressive neurodegenerative movement disorder primarily affecting the nigrostriatal dopaminergic system. The link between heightened activity of glycogen synthase kinase 3ß (GSK3ß) and neurodegene-rative processes has encouraged investigation into the potential disease-modifying effects of novel GSK3ß inhibitors in experimental models of PD. Therefore, the intriguing ability of several anesthetics to readily inhibit GSK3ß within the cortex and hippocampus led us to investigate the effects of brief isoflurane anesthesia on striatal GSK3ß signaling in naïve rats and in a rat model of early-stage PD. Deep but brief (20-min) isoflurane anesthesia exposure increased the phosphorylation of GSK3ß at the inhibitory Ser9 residue, and induced phosphorylation of AKTThr308 (protein kinase B; negative regulator of GSK3ß) in the striatum of naïve rats and rats with unilateral striatal 6-hydroxydopamine (6-OHDA) lesion. The 6-OHDA protocol produced gradual functional deficiency within the nigrostriatal pathway, reflected as a preference for using the limb ipsilateral to the lesioned striatum at 2 weeks post 6-OHDA. Interestingly, such motor impairment was not observed in animals exposed to four consecutive isoflurane treatments (20-min anesthesia every 48 h; treatments started 7 days after 6-OHDA delivery). However, isoflurane had no effect on striatal or nigral tyrosine hydroxylase (a marker of dopaminergic neurons) protein levels. This brief report provides promising results regarding the therapeutic potential and neurobiological mechanisms of anesthetics in experimental models of PD and guides development of novel disease-modifying therapies.
Subject(s)
Anesthesia/adverse effects , Corpus Striatum/metabolism , Isoflurane/pharmacology , Neuroprotective Agents/pharmacology , Parkinson Disease/metabolism , Signal Transduction/drug effects , Substantia Nigra/metabolism , Animals , Cyclic AMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Dopaminergic Neurons/drug effects , Glycogen Synthase Kinase 3/metabolism , Isoflurane/administration & dosage , Male , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Oxidopamine/pharmacology , Parkinson Disease/pathology , Rats, WistarABSTRACT
Many mechanisms have been proposed to explain acute antidepressant drug-induced activation of TrkB neurotrophin receptors, but several questions remain. In a series of pharmacological experiments, we observed that TrkB activation induced by antidepressants and several other drugs correlated with sedation, and most importantly, coinciding hypothermia. Untargeted metabolomics of pharmacologically dissimilar TrkB activating treatments revealed effects on shared bioenergetic targets involved in adenosine triphosphate (ATP) breakdown and synthesis, demonstrating a common perturbation in metabolic activity. Both activation of TrkB signaling and hypothermia were recapitulated by administration of inhibitors of glucose and lipid metabolism, supporting a close relationship between metabolic inhibition and neurotrophic signaling. Drug-induced TrkB phosphorylation was independent of electroencephalography slow-wave activity and remained unaltered in knock-in mice with the brain-derived neurotrophic factor (BDNF) Val66Met allele, which have impaired activity-dependent BDNF release, alluding to an activation mechanism independent from BDNF and neuronal activity. Instead, we demonstrated that the active maintenance of body temperature prevents activation of TrkB and other targets associated with antidepressants, including p70S6 kinase downstream of the mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3ß (GSK3ß). Increased TrkB, GSK3ß, and p70S6K phosphorylation was also observed during recovery sleep following sleep deprivation, when a physiological temperature drop is known to occur. Our results suggest that the changes in bioenergetics and thermoregulation are causally connected to TrkB activation and may act as physiological regulators of signaling processes involved in neuronal plasticity.
Subject(s)
Brain-Derived Neurotrophic Factor , Hypothermia , Animals , Mice , Antidepressive Agents/pharmacology , Brain-Derived Neurotrophic Factor/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Mammals/metabolism , Receptor, trkB/metabolism , Signal TransductionABSTRACT
Post-ictal emergence of slow wave EEG (electroencephalogram) activity and burst-suppression has been associated with the therapeutic effects of the electroconvulsive therapy (ECT), indicating that mere "cerebral silence" may elicit antidepressant actions. Indeed, brief exposures to burst-suppressing anesthesia has been reported to elicit antidepressant effects in a subset of patients, and produce behavioral and molecular alterations, such as increased expression of brain-derived neurotrophic factor (BDNF), connected with antidepressant responses in rodents. Here, we have further tested the cerebral silence hypothesis by determining whether repeated exposures to isoflurane anesthesia reduce depressive-like symptoms or influence BDNF expression in male Wistar outbred rats (Crl:WI(Han)) subjected to chronic mild stress (CMS), a model which is responsive to repeated electroconvulsive shocks (ECS, a model of ECT). Stress-susceptible, stress-resilient, and unstressed rats were exposed to 5 doses of isoflurane over a 15-day time period, with administrations occurring every third day. Isoflurane dosing is known to reliably produce rapid EEG burst-suppression (4% induction, 2% maintenance; 15 min). Antidepressant and anxiolytic effects of isoflurane were assessed after the first, third, and fifth drug exposure by measuring sucrose consumption, as well as performance on the open field and the elevated plus maze tasks. Tissue samples from the medial prefrontal cortex and hippocampus were collected, and levels of BDNF (brain-derived neurotrophic factor) protein were assessed. We find that isoflurane anesthesia had no impact on the behavior of stress-resilient or anhedonic rats in selected tests; findings which were consistent-perhaps inherently related-with unchanged levels of BDNF.
Subject(s)
Antidepressive Agents/pharmacology , Depressive Disorder/prevention & control , Isoflurane/pharmacology , Stress, Psychological/prevention & control , Anesthetics, Inhalation , Animals , Brain-Derived Neurotrophic Factor/metabolism , Depressive Disorder/etiology , Depressive Disorder/physiopathology , Disease Models, Animal , Electroconvulsive Therapy/methods , Electroencephalography , Electroshock/adverse effects , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/physiopathology , Humans , Isoflurane/administration & dosage , Male , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , Prefrontal Cortex/physiopathology , Rats, Wistar , Stress, Psychological/etiology , Stress, Psychological/physiopathologyABSTRACT
Dysregulation of the PI3K/Akt/mTOR pathway has been implicated in several brain disorders, including epilepsy. Rapamycin and similar compounds inhibit mTOR. complex 1 and have been reported to decrease seizures, delay seizure development, or prevent epileptogenesis in different animal models of genetic or acquired epilepsies. However, data for acquired epilepsy are inconsistent, which, at least in part, may be due to the poor brain penetration and long brain persistence of rapamycin and the fact that it blocks only one of the two cellular mTOR complexes. Here we examined the antiepileptogenic or disease-modifying effects of two novel, brain-permeable and well tolerated 1,3,5-triazine derivatives, the ATP-competitive mTORC1/2 inhibitor PQR620 and the dual pan-PI3K/mTORC1/2 inhibitor PQR530 in the intrahippocampal kainate mouse model, in which spontaneous seizures develop after status epilepticus (SE). Following kainate injection, the two compounds were administered over 2â¯weeksâ¯at doses previously been shown to block mTORC1/2 or PI3K/mTORC1/2 in the mouse brain. When spontaneous seizures were recorded by continuous (24/7) video-EEG recording starting 6 weeks after termination of treatment, no effects on incidence or frequency of seizures were observed. Drug treatment suppressed the epilepsy-induced activation of the PI3K/Akt/mTOR pathway in the hippocampus, but granule cell dispersion in the dentate gyrus was not prevented. When epilepsy-associated behavioral alterations were determined 12-14 weeks after kainate, mice pretreated with PQR620 or PQR530 exhibited reduced anxiety-related behavior in the light-dark box, indicating a disease-modifying effect. Overall, the data indicate that mTORC1/C2 or PI3K/mTORC1/C2 inhibition may not be an antiepileptogenic strategy for SE-induced epilepsy.
Subject(s)
Azabicyclo Compounds/pharmacology , Epilepsy, Temporal Lobe/prevention & control , Hippocampus/drug effects , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Triazines/pharmacology , Animals , Anxiety , Behavior, Animal/drug effects , Dentate Gyrus/drug effects , Dentate Gyrus/metabolism , Disease Models, Animal , Electroencephalography , Enzyme Inhibitors/pharmacology , Epilepsy, Temporal Lobe/chemically induced , Epilepsy, Temporal Lobe/etiology , Excitatory Amino Acid Agonists/toxicity , Hippocampus/metabolism , Kainic Acid/toxicity , Male , Mice , Phosphatidylinositol 3-Kinases , Seizures , Signal Transduction , Status Epilepticus/chemically induced , Status Epilepticus/complicationsABSTRACT
Mechanistic target of rapamycin (mTOR) regulates cell proliferation, growth and survival, and is activated in cancer and neurological disorders, including epilepsy. The rapamycin derivative ("rapalog") everolimus, which allosterically inhibits the mTOR pathway, is approved for the treatment of partial epilepsy with spontaneous recurrent seizures (SRS) in individuals with tuberous sclerosis complex (TSC). In contrast to the efficacy in TSC, the efficacy of rapalogs on SRS in other types of epilepsy is equivocal. Furthermore, rapalogs only poorly penetrate into the brain and are associated with peripheral adverse effects, which may compromise their therapeutic efficacy. Here we compare the antiseizure efficacy of two novel, brain-permeable ATP-competitive and selective mTORC1/2 inhibitors, PQR620 and PQR626, and the selective dual pan-PI3K/mTORC1/2 inhibitor PQR530 in two mouse models of chronic epilepsy with SRS, the intrahippocampal kainate (IHK) mouse model of acquired temporal lobe epilepsy and Tsc1GFAP CKO mice, a well-characterized mouse model of epilepsy in TSC. During prolonged treatment of IHK mice with rapamycin, everolimus, PQR620, PQR626, or PQR530; only PQR620 exerted a transient antiseizure effect on SRS, at well tolerated doses whereas the other compounds were ineffective. In contrast, all of the examined compounds markedly suppressed SRS in Tsc1GFAP CKO mice during chronic treatment at well tolerated doses. Thus, against our expectation, no clear differences in antiseizure efficacy were found across the three classes of mTOR inhibitors examined in mouse models of genetic and acquired epilepsies. The main advantage of the novel 1,3,5-triazine derivatives is their excellent tolerability compared to rapalogs, which would favor their development as new therapies for TORopathies such as TSC.
Subject(s)
Epilepsies, Partial/drug therapy , Everolimus/therapeutic use , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Sirolimus/therapeutic use , Tuberous Sclerosis/drug therapy , Animals , Disease Models, Animal , Epilepsies, Partial/physiopathology , Everolimus/pharmacology , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/therapeutic use , Male , Mice , Mice, Knockout , Treatment Outcome , Tuberous Sclerosis/physiopathologyABSTRACT
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.
Subject(s)
Acetylcholine/metabolism , Epilepsy, Temporal Lobe/metabolism , Status Epilepticus/metabolism , Acetylcholine/analysis , Acetylcholine/pharmacology , Amino Acids/analysis , Animals , Aspartic Acid/analysis , Disease Models, Animal , Electroencephalography/methods , Epilepsy/metabolism , Epilepsy, Temporal Lobe/physiopathology , Female , Glutamic Acid/analysis , Hippocampus/metabolism , Lysine/analysis , Microdialysis/methods , Neurotransmitter Agents/analysis , Neurotransmitter Agents/pharmacology , Pilocarpine , Rats , Scopolamine , Seizures/metabolism , Status Epilepticus/chemically induced , Status Epilepticus/physiopathology , Temporal Lobe/metabolismABSTRACT
OBJECTIVES: Deep burst-suppressing isoflurane anesthesia regulates signaling pathways connected with antidepressant responses in the rodent brain: activation of TrkB neurotrophin receptor and inhibition of GSK3ß kinase (glycogen synthase kinase 3ß). The main objective of this study was to investigate whether EEG (electroencephalogram) burst suppression correlates with these intriguing molecular alterations induced by isoflurane. METHODS: Adult male mice pre-implanted with EEG recording electrodes were subjected to varying concentrations of isoflurane (1.0-2.0% ad 20 min) after which medial prefrontal cortex samples were collected for molecular analyses, and the data retrospectively correlated to EEG (+/- burst suppression). RESULTS: Isoflurane dose-dependently increased phosphorylation of TrkBY816, CREBS133 (cAMP response element binding protein), GSK3ßS9 and p70S6kT412/S424. The time spent in burst suppression mode varied considerably between individual animals. Notably, a subset of animals subjected to 1.0-1.5% isoflurane showed no burst suppression. While p-GSK3ßS9, p-CREBS133 and p-p70S6kT412/S424 levels were increased in the samples obtained also from these animals, p-TrkBY816 levels remained unaltered. CONCLUSIONS: Isoflurane dose-dependently regulates TrkB and GSK3ß signaling and dosing associated with therapeutic outcomes in depressed patients produces most prominent effects.
Subject(s)
Anesthetics, Inhalation/administration & dosage , Antidepressive Agents/administration & dosage , Glycogen Synthase Kinase 3 beta/metabolism , Isoflurane/administration & dosage , Membrane Glycoproteins/metabolism , Prefrontal Cortex , Protein-Tyrosine Kinases/metabolism , Animals , Dose-Response Relationship, Drug , Electroencephalography , Male , Mice, Inbred C57BL , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , Prefrontal Cortex/physiopathology , Signal Transduction/drug effectsABSTRACT
Subanesthetic rather than anesthetic doses are thought to bring the rapid antidepressant effects of the NMDAR (N-methyl-d-aspartate receptor) antagonist ketamine. Among molecular mechanisms, activation of BDNF receptor TrkB along with the inhibition of GSK3ß (glycogen synthase kinase 3ß) are considered as critical molecular level determinants for ketamine's antidepressant effects. Hydroxynorketamines (2R,6R)-HNK and (2S,6S)-HNK), non-anesthetic metabolites of ketamine, have been proposed to govern the therapeutic effects of ketamine through a mechanism not involving NMDARs. However, we have shown that nitrous oxide, another NMDAR blocking anesthetic and a putative rapid-acting antidepressant, evokes TrkB-GSK3ß signaling alterations during rebound slow EEG (electroencephalogram) oscillations. We investigated here the acute effects of ketamine, 6,6-d2-ketamine (a ketamine analogue resistant to metabolism) and cis-HNK that contains (2R,6R) and (2S,6S) enantiomers in 1:1 ratio, on TrkB-GSK3ß signaling and concomitant electroencephalographic (EEG) alterations in the adult mouse cortex. Ketamine dose-dependently increased slow oscillations and phosphorylations of TrkBY816 and GSK3ßS9 in crude brain homogenates (i.e. sedative/anesthetic doses (>50â¯mg/kg, i.p.) produced more prominent effects than a subanesthetic dose (10â¯mg/kg, i.p.)). Similar, albeit less obvious, effects were seen in crude synaptosomes. A sedative dose of 6,6-d2-ketamine (100â¯mg/kg, i.p.) recapitulated the effects of ketamine on TrkB and GSK3ß phosphorylation while cis-HNK at a dose of 20â¯mg/kg produced negligible acute effects on TrkB-GSK3ß signaling or slow oscillations. These findings suggest that the acute effects of ketamine on TrkB-GSK3ß signaling are by no means restricted to subanesthetic (i.e. antidepressant) doses and that cis-HNK is not responsible for these effects.
Subject(s)
Glycogen Synthase Kinase 3 beta/metabolism , Hypnotics and Sedatives/pharmacology , Ketamine/analogs & derivatives , Ketamine/pharmacology , Membrane Glycoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Brain/metabolism , Brain Waves/drug effects , Dose-Response Relationship, Drug , Electroencephalography , Male , Mice , Phosphorylation/drug effects , Signal Transduction , Synaptosomes/metabolismABSTRACT
Rapid antidepressant effects of ketamine become most evident when its psychotomimetic effects subside, but the neurobiological basis of this "lag" remains unclear. Laughing gas (N2O), another NMDA-R (N-methyl-D-aspartate receptor) blocker, has been reported to bring antidepressant effects rapidly upon drug discontinuation. We took advantage of the exceptional pharmacokinetic properties of N2O to investigate EEG (electroencephalogram) alterations and molecular determinants of antidepressant actions during and immediately after NMDA-R blockade. Effects of the drugs on brain activity were investigated in C57BL/6 mice using quantitative EEG recordings. Western blot and qPCR were used for molecular analyses. Learned helplessness (LH) was used to assess antidepressant-like behavior. Immediate-early genes (e.g., bdnf) and phosphorylation of mitogen-activated protein kinase-markers of neuronal excitability-were upregulated during N2O exposure. Notably, phosphorylation of BDNF receptor TrkB and GSK3ß (glycogen synthase kinase 3ß) became regulated only gradually upon N2O discontinuation, during a brain state dominated by slow EEG activity. Subanesthetic ketamine and flurothyl-induced convulsions (reminiscent of electroconvulsive therapy) also evoked slow oscillations when their acute pharmacological effects subsided. The correlation between ongoing slow EEG oscillations and TrkB-GSK3ß signaling was further strengthened utilizing medetomidine, a hypnotic-sedative agent that facilitates slow oscillations directly through the activation of α2-adrenergic autoreceptors. Medetomidine did not, however, facilitate markers of neuronal excitability or produce antidepressant-like behavioral changes in LH. Our results support a hypothesis that transient cortical excitability and the subsequent regulation of TrkB and GSK3ß signaling during homeostatic emergence of slow oscillations are critical components for rapid antidepressant responses.
Subject(s)
Antidepressive Agents/pharmacology , Cerebral Cortex/metabolism , Electroencephalography , Neurons/metabolism , Receptor, trkB/metabolism , Signal Transduction , Anesthetics/pharmacology , Animals , Biomarkers/metabolism , Cerebral Cortex/drug effects , Dose-Response Relationship, Drug , Flurothyl/pharmacology , Glycogen Synthase Kinase 3 beta/metabolism , Homeostasis/drug effects , Ketamine/pharmacology , Medetomidine/pharmacology , Mice, Inbred C57BL , Neurons/drug effects , Nitrous Oxide/pharmacology , Signal Transduction/drug effects , Up-Regulation/drug effectsABSTRACT
Although electroconvulsive therapy (ECT) is among the most effective treatment options for pharmacoresistant major depressive disorder (MDD), some patients still remain refractory to standard ECT practise. Thus, there is a need for markers reliably predicting ECT non/response. In our study, we have taken a novel translational approach for discovering potential biomarkers for the prediction of ECT response. Our hypothesis was that the promoter methylation of p11, a multifunctional protein involved in both depressive-like states and antidepressant treatment responses, is differently regulated in ECT responders vs. nonresponders and thus be a putative biomarker of ECT response. The chronic mild stress model of MDD was adapted with the aim to obtain rats that are resistant to conventional antidepressant drugs (citalopram). Subsequently, electroconvulsive stimulation (ECS) was used to select responders and nonresponders, and compare p11 expression and promoter methylation. In the rat experiments we found that the gene promoter methylation and expression of p11 significantly correlate with the antidepressant effect of ECS. Next, we investigated the predictive properties of p11 promoter methylation in two clinical cohorts of patients with pharmacoresistant MDD. In a proof-of-concept clinical trial in 11 patients with refractory MDD, higher p11 promoter methylation was found in responders to ECT. This finding was replicated in an independent sample of 65 patients with pharmacoresistant MDD. This translational study successfully validated the first biomarker reliably predicting the responsiveness to ECT. Prescreening of this biomarker could help to identify patients eligible for first-line ECT treatment and also help to develop novel antidepressant treatment procedures for depressed patients resistant to all currently approved antidepressant treatments.
Subject(s)
Cell-Penetrating Peptides/genetics , Citalopram/therapeutic use , DNA Methylation , Depressive Disorder, Major/therapy , Electroconvulsive Therapy , Promoter Regions, Genetic , Adult , Aged , Animals , Antidepressive Agents/therapeutic use , Biomarkers/analysis , Depressive Disorder, Major/genetics , Disease Models, Animal , Female , Humans , Male , Middle Aged , Proof of Concept Study , Proteomics , Rats , Rats, Wistar , Treatment OutcomeABSTRACT
Outbred rat lines such as Wistar rats are commonly used for models of depressive disorders. Such rats arise from random mating schedules. Hence, genetic drift occurs in outbred populations which could lead to genotypic and phenotypic heterogeneity between rats from different vendors. Additionally, vendor specific rearing conditions could contribute to intrastrain variability. In the present study differences in behavioral responses to the chronic mild stress (CMS) model of depression within Wistar rat strains from different vendors are described. DNA methylation studies and mRNA expression analysis of p11 revealed that the behavioral differences between the substrains are reflected at the epigenetic and genetic level. The results suggest that there are breeder-dependent differences in vulnerability to stress in the CMS model of depression, which might bear on the validity of the model and contribute to contradictory findings and difficulties of replication between laboratories. P11 mRNA expression seems to be differently regulated depending on the quality of the stress response evoked by CMS exposure.
Subject(s)
Annexin A2/genetics , Annexin A2/metabolism , Behavior, Animal , Epigenesis, Genetic , Rats, Wistar , S100 Proteins/genetics , S100 Proteins/metabolism , Stress, Psychological/genetics , Stress, Psychological/physiopathology , Animals , Animals, Outbred Strains , Biomedical Research/economics , Commerce , Depressive Disorder/genetics , Depressive Disorder/physiopathology , Disease Models, Animal , Genetic Drift , Genetic Predisposition to Disease , Hippocampus/metabolism , Industry , Male , Prefrontal Cortex/metabolism , Promoter Regions, Genetic , RNA, Messenger/metabolism , Rats, Wistar/genetics , Rats, Wistar/psychology , Species SpecificityABSTRACT
In patients with medical-refractory schizophrenia electroconvulsive therapy (ECT), i.e., the induction of therapeutic seizures via cortical surface electrodes, is effectively used. Electroconvulsive stimulation (ECS) in rodents simulates ECT in humans and is applied to investigate the mechanisms underlying this treatment. Experimentally-induced reduced prepulse inhibition (PPI) of the acoustic startle response (ASR), i.e., the reduction of the startle response to an intense acoustic stimulus when this stimulus is shortly preceded by a weaker not-startling stimulus, serves as an endophenotype for neuropsychiatric disorders that are accompanied by disturbed sensorimotor gating, such as schizophrenia. Here we used rats selectively bred for high and low PPI to evaluate whether bifrontal cortical ECS would affect PPI. For this purpose, cortical screw electrodes were stereotactically implanted above the frontal cortex. After recovery ECS was applied for five consecutive days with stimuli of 1 ms pulse-width, 100 pulses/s, 1 s duration, ranging from 5.5 mA to 10 mA. PPI of ASR was measured one day before ECS, and on days 1, 7, and 14 after the last ECS. In rats with breeding-induced low PPI ECS increased PPI one week after stimulation. In contrast, ECS decreased PPI in rats with high PPI on the first day after stimulation. The reaction to the startle impulse was reduced by ECS without difference between groups. This work provides evidence that rats with breeding-induced high or low PPI could be used to further investigate the underlying mechanisms of ECT in neuropsychiatric disorders with disturbed sensorimotor gating like schizophrenia.
Subject(s)
Cerebral Cortex/physiology , Prepulse Inhibition/physiology , Reflex, Startle/physiology , Sensory Gating/physiology , Acoustic Stimulation , Animals , Electric Stimulation , Male , Prepulse Inhibition/genetics , Rats , Rats, Wistar , Reflex, Startle/genetics , Sensory Gating/geneticsABSTRACT
Anesthetics are widely used in medical practice and experimental research, yet the neurobiological basis governing their effects remains obscure. We have here used quantitative phosphoproteomics to investigate the protein phosphorylation changes produced by a 30 min isoflurane anesthesia in the adult mouse hippocampus. Altogether 318 phosphorylation alterations in total of 237 proteins between sham and isoflurane anesthesia were identified. Many of the hit proteins represent primary pharmacological targets of anesthetics. However, findings also enlighten the role of several other proteins-implicated in various biological processes including neuronal excitability, brain energy homeostasis, synaptic plasticity and transmission, and microtubule function-as putative (secondary) targets of anesthetics. In particular, isoflurane increases glycogen synthase kinase-3ß (GSK3ß) phosphorylation at the inhibitory Ser(9) residue and regulates the phosphorylation of multiple proteins downstream and upstream of this promiscuous kinase that regulate diverse biological functions. Along with confirmatory Western blot data for GSK3ß and p44/42-MAPK (mitogen-activated protein kinase; reduced phosphorylation of the activation loop), we observed increased phosphorylation of microtubule-associated protein 2 (MAP2) on residues (Thr(1620,1623)) that have been shown to render its dissociation from microtubules and alterations in microtubule stability. We further demonstrate that diverse anesthetics (sevoflurane, urethane, ketamine) produce essentially similar phosphorylation changes on GSK3ß, p44/p42-MAPK, and MAP2 as observed with isoflurane. Altogether our study demonstrates the potential of quantitative phosphoproteomics to study the mechanisms of anesthetics (and other drugs) in the mammalian brain and reveals how already a relatively brief anesthesia produces pronounced phosphorylation changes in multiple proteins in the central nervous system.
Subject(s)
Anesthetics, Inhalation/pharmacology , Hippocampus/drug effects , Isoflurane/pharmacology , Microtubules/metabolism , Neuronal Plasticity/drug effects , Anesthesia/methods , Animals , Male , Methyl Ethers/pharmacology , Mice, Inbred C57BL , Microtubule-Associated Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , SevofluraneABSTRACT
Electroconvulsive therapy is the most effective therapy for major depressive disorder (MDD). The remission rate is above 50% in previously pharmacoresistant patients but the mechanisms of action are not fully understood. Electroconvulsive stimulation (ECS) in rodents mimics antidepressant electroconvulsive therapy (ECT) in humans and is widely used to investigate the underlying mechanisms of ECT. For the translational value of findings in animal models it is essential to establish models with the highest construct, face and predictive validity possible. The commonly used model for ECT in rodents does not meet the demand for high construct validity. For ECT, cortical surface electrodes are used to induce therapeutic seizures whereas ECS in rodents is exclusively performed by auricular or corneal electrodes. However, the stimulation site has a major impact on the type and spread of the induced seizure activity and its antidepressant effect. We propose a method in which ECS is performed by screw electrodes placed above the motor cortex of rats to closely simulate the clinical situation and thereby increase the construct validity of the model. Cortical ECS in rats induced reliably seizures comparable to human ECT. Cortical ECS was more effective than auricular ECS to reduce immobility in the forced swim test. Importantly, auricular stimulation had a negative influence on the general health condition of the rats with signs of fear during the stimulation sessions. These results suggest that auricular ECS in rats is not a suitable ECT model. Cortical ECS in rats promises to be a valid method to mimic ECT.
Subject(s)
Depressive Disorder, Major/therapy , Electroconvulsive Therapy/methods , Animals , Cerebral Cortex/physiopathology , Disease Models, Animal , Electroencephalography , Immobility Response, Tonic/physiology , Rats , Rats, Wistar , Reproducibility of Results , Seizures/physiopathology , Swimming/psychology , Translational Research, BiomedicalABSTRACT
Several closely related strains of the thermophilic bacterium Dictyoglomus have been isolated from various hot springs on the Philippine archipelago. These strains as well as Dictyoglomus thermophilum H-6-12 were analyzed in view of the structure-functional relationships of the cell envelopes. All envelopes of Dictyoglomus strains show several peculiar features that are apparently either unique for the genus or common for other phylogenetically related Thermotogales. The filamentous cells develop pili at the cell poles, guided by large columnar protein assemblies that traverse the periplasm. Filamentous protein complexes span the periplasmic space at the longitudinal sides of the cell. By the end of the exponential growth phase, Dictyoglomus strains form multicellular aggregates ("rotund bodies") inside a compartment surrounded by a single, continuous outer envelope. The formation of these rotund bodies which are also found in some other deeply branching thermophilic phyla (Thermotoga, Thermus) was studied in detail. The transition between unicellular and multicellular lifestyle can be explained by the partial detachment of a protoplast from the outer envelope during cell division. When the outer envelope is partially detached from the protoplast, mechanical forces generated by protoplast elongation may drive cell rearrangement of daughter cells inside the compartment. During the following rounds of cell division, the overall shape of the compartment changes from spindle-like to globular geometry. Analysis of subcellular fractions of Dictyoglomus cells shows that glucan hydrolases are associated with the compartment. This feature is discussed in view of the multicellular life style of Dictyoglomus.