A random mutagenesis screen enriched for missense mutations in bacterial effector proteins.

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.

Random Cycle Loss and Its Application to Voice Conversion.

Speech disentanglement aims to decompose independent causal factors of speech signals into separate codes. Perfect disentanglement benefits to a broad range of speech processing tasks. This paper presents a simple but effective disentanglement approach based on cycle consistency loss and random factor substitution. This leads to a novel random cycle (RC) loss that enforces analysis-and-resynthesis consistency, a main principle of reductionism. We theoretically demonstrate that the proposed RC loss can achieve independent codes if well optimized, which in turn leads to superior disentanglement when combined with information bottleneck (IB). Extensive simulation experiments were conducted to understand the properties of the RC loss, and experimental results on voice conversion further demonstrate the practical merit of the proposal. Source code and audio samples can be found on the webpage http://rc.cslt.org.

Do 'Surgeon Champions' and High-Volume Surgeons Have Lower Rates of Periprosthetic Femur Fracture? Perspective From a State-Wide Quality Improvement Registry.

BACKGROUND: Recently, a state-wide registry identified fracture as a major cause of total hip arthroplasty revision. There were 52.8% of revisions occurring within 6 months (fracture leading cause). Registry sites have a 'surgeon champion' who acts as liaison and advocate. This study evaluated the effect of surgeon volume and role of 'surgeon champion' on fracture rates. METHODS: There were 95,948 cases from 2012 to 2019 queried with peri-implant femoral fractures identified (within 6 months). Funnel plots were generated to compare individual surgeon-specific fracture rates. Surgeons who had a fracture rate below the confidence interval were labeled 'green' (lower than mean), within were 'yellow' (no difference), and above were 'red' (significantly higher). RESULTS: For all surgeons, 19.6% were red, 72.1% yellow, and 8.3% green. There were 17.2% 'surgeon champions' and 6.2% 'nonchampions' that were green (P = .01), while 20.7 and 19.3% were red (P = .82). There was a significant association between volume and performance (P < .01). No surgeons in the lower two quartiles (<84; 84 to 180 cases), while 4 and 29% of higher-volume surgeons (181 to 404; >404 cases) were green. There was no statistical difference in red status by volume (P = .53). CONCLUSION: 'Surgeon champions' and high-volume surgeons were more likely to be high performers but not less likely to be low performers. Active involvement in quality improvement and/or high volume was associated with better outcomes but did not impart complication immunity. 'Green' surgeons should mentor colleagues to help reduce fractures by re-evaluating modifiable factors. Analyzing outcomes to promote quality and decrease complications is paramount.

Association between platelet indices and risk of preeclampsia in pregnant women.

We aimed to examine the association between the platelet indices and the risk of preeclampsia (PE) at different gestational weeks (GW) to explore the feasibility of early prediction of PE with these indices. About 7314 normotensive pregnant women and 396 PE patients were included and platelet indices, including platelet count (PC), plateletcrit (PCT), platelet distribution width (PDW), mean platelet volume (MPV) at different gestational weeks (1-12, 13-28, 29-32, 33-36 and 37-41 GW) were compared in two statistical methods. Patients with PE tended to have higher means of PC, PCT, PDW and MPV than normal pregnant women at early stage of pregnancy. The odds of PE were significantly increased with the increase of PC, PCT, PDW and MPV both at 13-28 GW and 29-32 GW, which indicated that increased values of PC, PCT, PDW and MPV at 13-32 GW were associated with greater subsequent risk of preeclampsia. Increased PC, PCT, PDW and MPV may have potential to predict preeclampsia before the disease onset.Impact StatementWhat is already known on this subject? Previous studies indicated that preeclampsia patients may have decreased platelet count (PC), plateletcrit (PCT) and increased platelet distribution width (PDW) and mean platelet volume (MPV). Increased PDW and MPV or decreased PC/MPV may have predictive values for PE.What do the results of this study add? The discrepancy with previous studies lay in the increased values of PC and PCT in PE patients at early stage of pregnancy. The study indicated that increased PC, PCT, PDW and MPV may have potential to predict preeclampsia far ahead of the disease onset. The results may reflect the abnormal turnover of platelets in PE patients.What are the implications of these findings for clinical practice and/or further research? These findings may help to guide early interventions before progress to overt preeclampsia by predicting onset of preeclampsia via easily available platelet indices in early weeks of gestation, which is especially valuable in areas lacking medical resources. The inconsistency with previous studies can facilitate researchers to further explore the coagulation mechanism beneath preeclampsia and pay more attention to the dynamic changes of platelet indices and other coagulation indices during pregnancy.

Causes of Early Hip Revision Vary by Age and Gender: Analysis of Data From a Statewide Quality Registry.

BACKGROUND: While total hip arthroplasty (THA) is extremely successful, early failures do occur. The purpose of this study was to determine the cause of revision in specific patient demographic groups at 3 time points to potentially help decrease the revision risk. METHODS: Data for cases performed between 2012 and 2018 from a statewide, quality improvement arthroplasty registry were used. The database included 79,205 THA cases and 1,433 revisions with identified etiology (1,584 in total). All revisions performed at <5 years from the primary THA were reviewed. Six groups, men/women, <65, 65-75, and >75 years, were compared at revision time points <6 months, <1 year, and <5 years. RESULTS: There were obvious and significant differences between subgroups based on demographics and time points (P < .0001). Seven hundred and fifty-six (53%) of all revisions occurred within 6 months. The most common etiologies within 6 months (756 revisions) were fracture (316, 41.8%), dislocation/instability (194, 25.7%), and infection (98, 12.9%). At this early time point, the most common revision cause was fracture for all age/gender-stratified groups, ranging from 27.6% in young men to 60% in older women. Joint instability became the leading cause for revision after 1 year in all groups. CONCLUSION: This quality improvement project demonstrated clinically meaningful differences in the reason for THA revision between gender, age, and time from surgery. Strategies based on these data should be employed by surgeons to minimize the factors that lead to revision.

Cav 3.2 T-type calcium channel mutation influences kindling-induced thalamic neuronal firing patterns in Genetic Absence Epilepsy Rats From Strasbourg.

OBJECTIVE: Recent data indicate that amygdala kindling leads to significant changes in interictal neuronal firing patterns of thalamic reticular nucleus (TRN) neurons by decreasing the spontaneous firing rate and increasing burst firing in nonepileptic control (NEC) rats. Genetic Absence Epilepsy Rats From Strasbourg (GAERS) were resistant to these kindling-induced firing changes in TRN neurons, and are also resistant to the progression of kindling. We investigated whether a homozygous, missense, single nucleotide mutation (R1584P) in the Cav 3.2 T-type Ca2+ channel gene, which has been correlated with the expression of absence seizures in GAERS, influenced kindling progression and TRN firing patterns. METHODS: Double-crossed (GAERS vs NEC; F2) rats that were homozygous for the Cav 3.2 mutation (PP) and those negative for the mutation (RR) were implanted with a stimulating electrode in the amygdala. Rats received a total of 30 kindling stimulations at their afterdischarge threshold current twice daily, and kindling progression was evaluated. Thereafter, the extracellular neuronal activity of TRN neurons was recorded in vivo under neuroleptanesthesia to investigate the influence of Cav 3.2 mutation on TRN firing patterns. RESULTS: We found that the R1584P mutation did not affect kindling progression in F2 crosses (P = 0.78). However, it influenced kindling-induced neuronal firing of TRN neurons. After 30 stimulations, RR rats exhibited a lower firing rate and a higher percentage of burst firing compared to PP rats. The decrease in firing frequency was correlated with the increase in the amount of burst firing in RR rats (R2  = 0.497). SIGNIFICANCE: Our findings suggest that mutation in Cav 3.2 T-type Ca2+ channels may play a role in the resistance to kindling-induced changes in TRN neurons to a low-frequency and high-percentage bursting pattern seen in association with the convulsive stages of amygdala kindling, but is not in itself enough to explain the resistance to kindling progression observed in GAERS.

Neuropeptide Y affects thalamic reticular nucleus neuronal firing and network synchronization associated with suppression of spike-wave discharges.

OBJECTIVES: Neuropeptide Y (NPY) potently suppresses spike-wave discharges (SWDs) in a genetic rat model of absence epilepsy (GAERS), but the underlying neurophysiologic mechanisms are not clear. We therefore sought to determine the in vivo effects of NPY on neuronal firing in the cortico-thalamo-cortical network activity, known to play a critical role in the generation of SWDs in these rats. METHODS: NPY was administered intracerebroventricularly (ICV) or in separate experiments locally on the neurons of caudal thalamic reticular nucleus (NRT) by use of juxtacellular iontophoresis in triple-barrel electrodes in male GAERS aged 12-15 weeks, in vivo under neuroleptic anesthesia. Drug infusions and electroencephalography (EEG) monitoring were performed simultaneously with juxtacellular single neuronal recordings. Effect of NPY on electrically induced SWD induction threshold were also measured. RESULTS: NPY administration ICV led to a decrease in the total length of SWDs in EEG recordings. Both ICV administration and iontophoresis of NPY on NRT neurons led to an increase in interictal neuronal firing of NRT neurons. During ictal periods, ICV NPY administration reduced the number of thalamic action potentials per SWDs, as well as reduced waveform correlations between field potentials within the NRT and the cortical EEG. NPY administration ICV did not significantly alter the firing patterns of relay thalamic neurons interictally and cortical neurons during ictal and interictal periods. In addition, SWD induction threshold in the S2 region of the cortex was significantly increased after NPY administration. SIGNIFICANCE: Our results show alterations in cortico-thalamo-cortical local and network properties following ICV administration of NPY, suggesting mechanisms of SWD suppression in GAERS. Cellular and network alteration of NRT activity, resulting from a direct action of NPY, may be a contributor to this effect.

The peroxisome proliferator activated receptor gamma agonist pioglitazone increases functional expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in human glioblastoma cells.

Glioma cells release glutamate through expression of system xc-, which exchanges intracellular glutamate for extracellular cysteine. Lack of the excitatory amino acid transporter 2 (EAAT2) expression maintains high extracellular glutamate levels in the glioma microenvironment, causing excitotoxicity to surrounding parenchyma. Not only does this contribute to the survival and proliferation of glioma cells, but is involved in the pathophysiology of tumour-associated epilepsy (TAE). We investigated the role of the peroxisome proliferator activated receptor gamma (PPARγ) agonist pioglitazone in modulating EAAT2 expression in glioma cells. We found that EAAT2 expression was increased in a dose dependent manner in both U87MG and U251MG glioma cells. Extracellular glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in both U87MG and U251MG cells at a concentration of ≥ 30 µM pioglitazone (p < 0.05). The PPARγ antagonist GW9662 inhibited the effect of pioglitazone on extracellular glutamate levels, indicating PPARγ dependence. In addition, pioglitazone significantly reduced cell viability of U87MG and U251MG cells at ≥ 30 µM and 100 µM (p < 0.05) respectively. GW9662 also significantly reduced viability of U87MG and U251MG cells with 10 µM and 30 µM (p < 0.05) respectively. The effect on viability was partially dependent on PPARγ activation in U87MG cells but not U251MG cells, whereby PPARγ blockade with GW9662 had a synergistic effect. We conclude that PPARγ agonists may be therapeutically beneficial in the treatment of gliomas and furthermore suggest a novel role for these agents in the treatment of tumour associated seizures through the reduction in extracellular glutamate.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models.

OBJECTIVE: Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. METHODS: Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. RESULTS: Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. SIGNIFICANCE: These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

The effect of amygdala kindling on neuronal firing patterns in the lateral thalamus in the GAERS model of absence epilepsy.

OBJECTIVE: The co-occurrence of absence and mesial temporal lobe epilepsy is rare in both humans and animal models. Consistent with this, rat models of absence epilepsy, including genetic absence epilepsy rats from Strasbourg (GAERS), are resistant to experimental temporal lobe epileptogenesis, in particular by amygdala kindling. Structures within the cortical-thalamocortical system are critically involved in the generation and maintenance of the electrographic spike-and-wave discharges (SWDs) that characterize absence seizures. Using in vivo electrophysiologic recordings, this study investigated the role of thalamocortical circuitry in the generalization of amygdala-kindling induced seizures in the GAERS and the nonepileptic control (NEC) strain of Wistar rats. METHODS: GAERS and NEC rats were implanted with a stimulating electrode in amygdala and stimulated at afterdischarge threshold twice daily to a maximum number of 30 stimulations. Thereafter extracellular single neuron recordings were performed in vivo under neuroleptanesthesia in the thalamocortical network. RESULTS: In NEC rats, amygdala kindling induced convulsive class V seizures and altered characteristics of neuronal activity in the thalamic reticular nucleus (TRN), in particular decreased firing rates and increased burst firing patterns. Less marked changes were seen in other regions examined: the ventroposteromedial nucleus of thalamus (VPM), the CA3 region of the hippocampus, and the deep layers (V/VI) of the cortex. GAERS did not progress beyond class II seizures, with a matched number of kindling stimulations, and the thalamic neuronal firing alterations observed in NEC rats were not seen. SIGNIFICANCE: These data suggest that the TRN plays an important role in kindling resistance in GAERS and is central to the control of secondary generalization of limbic seizures.

Acute effect of carbamazepine on corticothalamic 5-9-Hz and thalamocortical spindle (10-16-Hz) oscillations in the rat.

A major side effect of carbamazepine (CBZ), a drug used to treat neurological and neuropsychiatric disorders, is drowsiness, a state characterized by increased slow-wave oscillations with the emergence of sleep spindles in the electroencephalogram (EEG). We conducted cortical EEG and thalamic cellular recordings in freely moving or lightly anesthetized rats to explore the impact of CBZ within the intact corticothalamic (CT)-thalamocortical (TC) network, more specifically on CT 5-9-Hz and TC spindle (10-16-Hz) oscillations. Two to three successive 5-9-Hz waves were followed by a spindle in the cortical EEG. A single systemic injection of CBZ (20 mg/kg) induced a significant increase in the power of EEG 5-9-Hz oscillations and spindles. Intracellular recordings of glutamatergic TC neurons revealed 5-9-Hz depolarizing wave-hyperpolarizing wave sequences prolonged by robust, rhythmic spindle-frequency hyperpolarizing waves. This hybrid sequence occurred during a slow hyperpolarizing trough, and was at least 10 times more frequent under the CBZ condition than under the control condition. The hyperpolarizing waves reversed at approximately -70 mV, and became depolarizing when recorded with KCl-filled intracellular micropipettes, indicating that they were GABAA receptor-mediated potentials. In neurons of the GABAergic thalamic reticular nucleus, the principal source of TC GABAergic inputs, CBZ augmented both the number and the duration of sequences of rhythmic spindle-frequency bursts of action potentials. This indicates that these GABAergic neurons are responsible for the generation of at least the spindle-frequency hyperpolarizing waves in TC neurons. In conclusion, CBZ potentiates GABAA receptor-mediated TC spindle oscillations. Furthermore, we propose that CT 5-9-Hz waves can trigger TC spindles.

Enduring Effects of Early Life Stress on Firing Patterns of Hippocampal and Thalamocortical Neurons in Rats: Implications for Limbic Epilepsy.

Early life stress results in an enduring vulnerability to kindling-induced epileptogenesis in rats, but the underlying mechanisms are not well understood. Recent studies indicate the involvement of thalamocortical neuronal circuits in the progression of kindling epileptogenesis. Therefore, we sought to determine in vivo the effects of early life stress and amygdala kindling on the firing pattern of hippocampus as well as thalamic and cortical neurons. Eight week old male Wistar rats, previously exposed to maternal separation (MS) early life stress or early handling (EH), underwent amygdala kindling (or sham kindling). Once fully kindled, in vivo juxtacellular recordings in hippocampal, thalamic and cortical regions were performed under neuroleptic analgesia. In the thalamic reticular nucleus cells both kindling and MS independently lowered firing frequency and enhanced burst firing. Further, burst firing in the thalamic reticular nucleus was significantly increased in kindled MS rats compared to kindled EH rats (p<0.05). In addition, MS enhanced burst firing of hippocampal pyramidal neurons. Following a stimulation-induced seizure, somatosensory cortical neurons exhibited a more pronounced increase in burst firing in MS rats than in EH rats. These data demonstrate changes in firing patterns in thalamocortical and hippocampal regions resulting from both MS and amygdala kindling, which may reflect cellular changes underlying the enhanced vulnerability to kindling in rats that have been exposed to early life stress.

An in vivo technique for investigating electrophysiological effects of centrally administered drugs on single neurons and network behaviour.

Single neuronal juxtacellular recording with simultaneous cortical electroencephalogram (EEG) in whole-animal preparations in vivo has allowed the study of the behaviour of individual neurons in relation to whole brain activity. Data on single neuron firing, neural synchrony, network behaviour and their responses to pharmacological agents can be obtained with dual recordings. However, pharmacological effects on cellular and network activity during paired single-unit recordings have not been possible due to the difficulties in maintaining recordings of two cells for a prolonged period. Here, we describe a method of maintaining stable dual cell juxtacellular recordings from distinct brain regions, allowing the assessment of single unit activity before, during and after the intracerebroventricular (ICV) injection of drugs. Data collection using this technique allows correlation both between the two cells and with whole-brain EEG, and their responses to pharmacological interventions. This is particularly useful for the investigation of the effects of anti-epileptic drugs on animal models of epilepsy, where single unit activity of two cells from distinct regions can be correlated with each other and with whole-brain activity during pre-ictal, ictal and interictal states. We also describe standardised analytical methods of quantifying cell firing patterns, the rhythmicity of individual neurons and the synchronicity of firing between two neurons in ictal and interictal periods and their responses to drug exposure.

Rhythmic neuronal activity in S2 somatosensory and insular cortices contribute to the initiation of absence-related spike-and-wave discharges.

PURPOSE: The origin of bilateral synchronous spike-and-wave discharges (SWDs) that underlie absence seizures has been widely debated. Studies in genetic rodent models suggest that SWDs originate from a restricted region in the somatosensory cortex. The properties of this initiation site remain unknown. Our goal was to characterize the interictal, preictal and ictal neuronal activity in the primary and secondary cortical regions (S1, S2) and in the adjacent insular cortex (IC) in Genetic Absence Epilepsy Rats from Strasbourg (GAERS). METHODS: We performed electroencephalography (EEG) recordings in combination with multisite local field potential (LFP) and single cell juxtacellular recordings, and cortical electrical stimulations, in freely moving rats and those under neurolept-anesthesia. KEY FINDINGS: The onset of the SWDs was preceded by 5-9 Hz field potential oscillations, which were detected earlier in S2 and IC than in S1. Sustained SWDs could be triggered by a 2-s train of 7-Hz electrical stimuli at a lower current intensity in S2 than in S1. In S2 and IC, subsets of neurons displayed rhythmic firing (5-9 Hz) in between seizures. S2 and IC layers V and VI neurons fired during the same time window, whereas in S1 layer VI, neurons fired before layer V neurons. Just before the spike component of each SW complex, short-lasting high-frequency oscillations consistently occurred in IC ∼20 msec before S1. SIGNIFICANCE: Our findings demonstrate that the S2/IC cortical areas are a critical component of the macro-network that is responsible for the generation of absence-related SWDs.

Opposite effects of ketamine and deep brain stimulation on rat thalamocortical information processing.

Sensory and cognitive deficits are common in schizophrenia. They are associated with abnormal brain rhythms, including disturbances in γ frequency (30-80 Hz) oscillations (GFO) in cortex-related networks. However, the underlying anatomofunctional mechanisms remain elusive. Clinical and experimental evidence suggests that these deficits result from a hyporegulation of glutamate N-methyl-D-aspartate receptors. Here we modeled these deficits in rats with ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist and a translational psychotomimetic substance at subanesthetic doses. We tested the hypothesis that ketamine-induced sensory deficits involve an impairment of the ability of the thalamocortical (TC) system to discriminate the relevant information from the baseline activity. Furthermore, we wanted to assess whether ketamine disrupts synaptic plasticity in TC systems. We conducted multisite network recordings in the rat somatosensory TC system, natural stimulation of the vibrissae and high-frequency electrical stimulation (HFS) of the thalamus. A single systemic injection of ketamine increased the amount of baseline GFO, reduced the amplitude of the sensory-evoked TC response and decreased the power of the sensory-evoked GFO. Furthermore, cortical application of ketamine elicited local and distant increases in baseline GFO. The ketamine effects were transient. Unexpectedly, HFS of the TC pathway had opposite actions. In conclusion, ketamine and thalamic HFS have opposite effects on the ability of the somatosensory TC system to discriminate the sensory-evoked response from the baseline GFO during information processing. Investigating the link between the state and function of the TC system may conceptually be a key strategy to design innovative therapies against neuropsychiatric disorders.

Excitotoxic-mediated transcriptional decreases in HCN2 channel function increase network excitability in CA1.

Changes in the conductance of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel that mediates Ih are proposed to contribute to increased network excitability. Synchronous neuronal burst activity is a good reflection of network excitability and can be generated in isolated hippocampal slice cultures by removing Mg2+ from the extracellular fluid. We demonstrate that Ih contributes to this activity by increasing both the frequency and duration of bursting events. Changes in HCN channel function are also implicated in altered seizure susceptibility. Short-term application of kainic acid (KA) is known to initiate long lasting changes in neuronal networks that result in seizures, and in slice cultures was found to alter HCN mRNA levels in an isoform and hippocampal sub-region specific manner. These changes correlate with the ability of each sub-region to develop synchronous burst activity following KA that we have previously reported. Specifically, a loss of synchronous activity in the CA3 correlated with an increase in HCN2 mRNA levels that normalized concomitantly with the restoration of CA3 burst activity 7 days post insult. In contrast, in CA1 an increase in synchronous burst duration correlated with a reduction in HCN2 mRNA levels and both changes were still evident for 7 days post insult. Lamotrigine, known to increase Ih, reversed the impact of KA on burst duration in CA1 at both time-points linking a transcriptional reduction in HCN2 function to increased burst duration.

Oxcarbazepine, not its active metabolite, potentiates GABAA activation and aggravates absence seizures.

PURPOSE: Studies in genetic absence epileptic rats from Strasbourg (GAERS) indicate that enhancement of gamma aminobutyric acid (GABA(A)) receptor activity is a critical mechanism in the aggravation of seizures by carbamazepine (CBZ). We examined whether structural analogs of CBZ, oxcarbazepine (OXC), and its active metabolite, monohydroxy derivative (MHD), also potentiate GABA(A) receptor current and aggravate seizures. METHODS: In vitro studies in Xenopus oocytes compared the three drugs' effect on GABA(A) receptor currents. In vivo studies compared seizure activity in GAERS after intraperitoneal drug administration. RESULTS: OXC potentiated GABA(A) receptor current and aggravated seizures in GAERS, similarly to the effect of CBZ. Conversely, MHD showed only a minor potentiation of GABA(A) receptor current and did not aggravate seizures. DISCUSSION: A hydroxyl group at the C-10 position on the CBZ tricyclic structure in MHD reduces GABA(A) receptor potentiation and seizure aggravation. Reports of the aggravation of absence seizures in patients taking OXC may result from circulating unmetabolized OXC rather than MHD.

The mechanism of carbamazepine aggravation of absence seizures.

Carbamazepine (CBZ) aggravates many generalized seizures types, particularly absence seizures, but the mechanisms underlying this are poorly understood. GABA signaling within the reticular nucleus (Rt) and the ventrobasal complex (VB) of the thalamus is critical to the neurophysiology of absence seizures. The hypothesis that CBZ aggravates absence seizures by acting at the VB thalamus via a GABA(A) receptor-mediated mechanism was investigated in a genetic rat model, generalized absence epilepsy rats from Strasbourg (GAERS). Seizure activity was quantified by a 90-min electroencephalogram recording postdrug injection. Intracerebroventricular injections of CBZ (15 microg in 4 microl) resulted in seizure aggravation versus vehicle treatment, with a mean increase in seizure time of 40%. This indicates that CBZ acts directly, rather than via a metabolite, on the brain to aggravate seizures. Seizure aggravation also occurred following bilateral microinjection of CBZ (0.75 microg in 0.2 microl) into the VB (53%) but not following injection into the Rt (-9%). However, seizure aggravation was blocked when the GABA(A) receptor antagonist, bicuculline (BIC, 0.04 microg in 0.2 microl), was coinjected with CBZ into the VB. Injection of BIC alone (versus vehicle) into the VB also blocked seizure aggravation following systemic administration of CBZ (15 mg/kg i.p.). In vitro studies in Xenopus oocytes expressing recombinant GABA(A) receptors demonstrated that CBZ produced a dose-dependent potentiation of the GABA current at a physiological relevant concentration range (1-100 microM). These data demonstrate that CBZ acts at the VB thalamus to aggravate absence seizures in GAERS and that activation of GABA(A) receptors is critical to this effect.

Angiotensin-converting enzyme (ACE) interacts with dopaminergic mechanisms in the brain to modulate prepulse inhibition in mice.

A renin-angiotensin system, separate to that in the periphery, has been found in the brain. Angiotensin-converting enzyme (ACE) is crucial in the synthesis of angiotensin II, breakdown of bradykinin and the hydrolysis of several other neuropeptides such as enkephalin, substance P, dynorphin and neurotensin. Changes in the levels of ACE have been found in brains of schizophrenia patients, suggesting an involvement of ACE in the illness which awaits further investigation. Prepulse inhibition (PPI) has been suggested to be an operational measure of sensorimotor gating and is disrupted in patients with schizophrenia. We found that ACE knockout mice have increased startle responses but no differences in baseline PPI compared to wildtype controls. Treatment with the dopamine receptor agonist, apomorphine, or the dopamine-releasing drug, amphetamine, produced significant disruption of PPI in control mice but not in ACE knockout mice. Pretreatment with the ACE inhibitor, captopril, which itself did not affect PPI, caused a reduction in the effect of apomorphine on PPI, similar to that seen in the ACE knockout mice. These data suggest an important role of ACE substrates in modulating dopaminergic mechanisms involved in PPI. Further studies are needed to ascertain if angiotensin or other neuropeptides are involved in these interactions and to investigate the neurochemical mechanism behind these effects.