Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy

Patch–clamp recordings of CA1 interneurons and pyramidal cells were performed in hippocampal slices from kainate- or pilocarpine-treated rat models of temporal lobe epilepsy. We report that γ-aminobutyric acid (GABA)ergic inhibition in pyramidal neurons is still functional in temporal lobe epilepsy because: (i) the frequency of spontaneous GABAergic currents is similar to that of control and (ii) focal electrical stimulation of interneurons evokes a hyperpolarization that prevents the generation of action potentials. In paired recordings of interneurons and pyramidal cells, synchronous interictal activities were recorded. Furthermore, large network-driven GABAergic inhibitory postsynaptic currents were present in pyramidal cells during interictal discharges. The duration of these interictal discharges was increased by the GABA type A antagonist bicuculline. We conclude that GABAergic inhibition is still present and functional in these experimental models and that the principal defect of inhibition does not lie in a complete disconnection of GABAergic interneurons from their glutamatergic inputs.     

Inhibition of ceramide biosynthesis preserves photoreceptor structure and function in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetic disease causing progressive apoptotic death of photoreceptors and, ultimately, incurable blindness. Using the retinal degeneration 10 (rd10) mouse model of RP, we investigated the role of ceramide, a proapoptotic sphingolipid, in retinal degeneration. We also tested the possibility that photoreceptor loss can be slowed or blocked by interfering with the ceramide signaling pathway of apoptosis in vivo. Retinal ceramide levels increased in rd10 mice during the period of maximum photoreceptor death. Single intraocular injections of myriocin, a powerful inhibitor of serine palmitoyl-CoA transferase, the rate-limiting enzyme of ceramide biosynthesis, lowered retinal ceramide levels to normal values and rescued photoreceptors from apoptotic death. Noninvasive treatment was achieved using eye drops consisting of a suspension of solid lipid nanoparticles loaded with myriocin. Short-term noninvasive treatment lowered retinal ceramide in a manner similar to intraocular injections, indicating that nanoparticles functioned as a vector permitting transcorneal drug administration. Prolonged treatment (10-20 d) with solid lipid nanoparticles increased photoreceptor survival, preserved photoreceptor morphology, and extended the ability of the retina to respond to light as assessed by electroretinography. In conclusion, pharmacological targeting of ceramide biosynthesis slowed the progression of RP in a mouse model, and therefore may represent a therapeutic approach to treating this disease in humans. Transcorneal administration of drugs carried in solid lipid nanoparticles, as experimented in this study, may facilitate continuous, noninvasive treatment of patients with RP and other retinal pathologies.     

Impaired hippocampal rhythmogenesis in a mouse model of mesial temporal lobe epilepsy

Mesial temporal lobe epilepsy (mTLE) is one of the most common forms of epilepsy, characterized by hippocampal sclerosis and memory deficits. Injection of kainic acid (KA) into the dorsal hippocampus of mice reproduces major electrophysiological and histopathological characteristics of mTLE. In extracellular recordings from the morphologically intact ventral hippocampus of KA-injected epileptic mice, we found that theta-frequency oscillations were abolished, whereas gamma oscillations persisted both in vivo and in vitro. Whole-cell recordings further showed that oriens-lacunosum-moleculare (O-LM) interneurons, key players in the generation of theta rhythm, displayed marked changes in their intrinsic and synaptic properties. Hyperpolarization-activated mixed cation currents (Ih) were significantly reduced, resulting in an increase in the input resistance and a hyperpolarizing shift in the resting membrane potential. Additionally, the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) was increased, indicating a stronger excitatory input to these neurons. As a consequence, O-LM interneurons increased their firing rate from theta to gamma frequencies during induced network activity in acute slices from KA-injected mice. Thus, our physiological data together with network simulations suggest that changes in excitatory input and synaptic integration in O-LM interneurons lead to impaired rhythmogenesis in the hippocampus that in turn may underlie memory deficit.     

Syndecan-4 over-expression preserves cardiac function in a rat model of myocardial infarction

Syndecan-4 (synd4) is a heparan sulfate proteoglycan, involved in repair following tissue damage, through modulating neovascularization and inflammation. In acute myocardial infarction its myocardial expression is up-regulated in a time-dependent manner, and in synd4-deficient mice severe cardiac dysfunction and abnormal remodeling are observed following induction of myocardial infarction. Here we explored the therapeutic potential of sustained synd4 over-expression in the context of myocardial infarction. Adenovirus containing the synd4 gene (Ad-synd4), or corresponding control adenovirus (Ad-null), was administered intramyocardially in rats immediately after induction of myocardial infarction. Cardiac function was ascertained by echocardiography, hemodynamic assessment and brain natriuretic peptide level 28 days post-intervention. Hearts were excised for molecular and histological analyses at predetermined time points. We observed reduced mortality and improved cardiac function post-myocardial infarction in the Ad-synd4 as compared to the Ad-null group, with associated attenuation of cardiac remodeling, less myocyte loss and reduced fibrosis. Additionally, the Ad-synd4 group exhibited endothelial cell activation and increased angiogenesis and arteriogenesis in the myocardium. The Ad-synd4 group also showed evidence of reduced myocardial inflammation as compared with the Ad-null group, with reduced inflammatory cell (CD45+) and myofibroblast (α-SMA+) infiltration as well as suppressed collagen III deposition and iNOS expression. Our results suggest that sustained synd4 over-expression in the myocardium is of therapeutic benefit following experimental myocardial infarction, through inducing neovascularization, suppressing tissue inflammation and fibrosis, with resultant improvements in cardiac function and remodeling.     

Gene therapy prevents photoreceptor death and preserves retinal function in a Bardet-Biedl syndrome mouse model

Patients with Bardet-Biedl syndrome (BBS) experience severe retinal degeneration as a result of impaired photoreceptor transport processes that are not yet fully understood. To date, there is no effective treatment for BBS-associated retinal degeneration, and blindness is imminent by the second decade of life. Here we report the development of an adeno-associated viral (AAV) vector that rescues rhodopsin mislocalization, maintains nearly normal-appearing rod outer segments, and prevents photoreceptor death in the Bbs4-null mouse model. Analysis of the electroretinogram a-wave indicates that rescued rod cells are functionally indistinguishable from wild-type rods. These results demonstrate that gene therapy can prevent retinal degeneration in a mammalian BBS model.     

Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy

Mutations in the GABA(A) receptor gamma2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a gamma2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of gamma2(R43Q) was seen in heterozygous mice in the absence of any change in alpha1 subunit surface expression, ruling out a dominant-negative effect. GABA(A)-mediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.     

Long-term inhibition of dipeptidyl peptidase IV improves glucose tolerance and preserves islet function in mice

OBJECTIVES: Inhibitors of the glucagon-like peptide-1 (GLP-1)-degrading enzyme, dipeptidyl peptidase IV (DPPIV), are being explored in the treatment of diabetes. We examined the long-term influence of a selective, orally active inhibitor of DPPIV (NVP DPP728), in normal female C57BL/6J mice and such mice rendered glucose-intolerant and insulin-resistant by feeding a high-fat diet. DESIGN: In mice fed a standard diet (11% fat) or a high-fat diet (58% fat), NVP DPP728 (0.12 micromol/g body weight) was administered in the drinking water for an 8 week period. RESULTS: DPPIV inhibition reduced plasma DPPIV activity to 0.01+/-0.03 mU/ml vs 3.26+/-0.19 mU/ml in controls (P<0.001). Glucose tolerance after gastric glucose gavage, as judged by the area under the curve for plasma glucose levels over the 120 min study period, was increased after 8 weeks by NVP DPP728 in mice fed normal diet (P=0.029) and in mice fed a high-fat diet (P=0.036). This was accompanied by increased plasma levels of insulin and intact GLP-1. Glucose-stimulated insulin secretion from islets isolated from NVP DPP728-treated animals after 8 weeks of treatment was increased as compared with islets from control animals at 5.6, 8.3 and 11.1 mmol/l glucose both in mice fed normal diet and in mice fed a high-fat diet (both P<0.05). Islet insulin and glucagon immunocytochemistry revealed that NVP DPP728 did not affect the islet architecture. However, the expression of immunoreactive glucose transporter isoform-2 (GLUT-2) was increased by DPPIV inhibition, and in mice fed a high-fat diet, islet size was reduced after treatment with NVP DPP728 from 16.7+/-2.6 x 10(3) microm(2) in controls to 7.6+/-1.0 x 10(3) microm(2) (P=0.0019). CONCLUSION: Long-term DPPIV inhibition improves glucose tolerance in both normal and glucose-intolerant mice through improved islet function as judged by increased GLUT-2 expression, increased insulin secretion and protection from increased islet size in insulin resistance.     

Seizures and enhanced cortical GABAergic inhibition in two mouse models of human autosomal dominant nocturnal frontal lobe epilepsy

Selected mutations in the human alpha4 or beta2 neuronal nicotinic acetylcholine receptor subunit genes cosegregate with a partial epilepsy syndrome known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To examine possible mechanisms underlying this inherited epilepsy, we engineered two ADNFLE mutations (Chrna4(S252F) and Chrna4(+L264)) in mice. Heterozygous ADNFLE mutant mice show persistent, abnormal cortical electroencephalograms with prominent delta and theta frequencies, exhibit frequent spontaneous seizures, and show an increased sensitivity to the proconvulsant action of nicotine. Relative to WT, electrophysiological recordings from ADNFLE mouse layer II/III cortical pyramidal cells reveal a >20-fold increase in nicotine-evoked inhibitory postsynaptic currents with no effect on excitatory postsynaptic currents. i.p. injection of a subthreshold dose of picrotoxin, a use-dependent gamma-aminobutyric acid receptor antagonist, reduces cortical electroencephalogram delta power and transiently inhibits spontaneous seizure activity in ADNFLE mutant mice. Our studies suggest that the mechanism underlying ADNFLE seizures may involve inhibitory synchronization of cortical networks via activation of mutant alpha4-containing nicotinic acetylcholine receptors located on the presynaptic terminals and somatodendritic compartments of cortical GABAergic interneurons.     

Role of proinflammatory cytokines in the inhibition of hippocampal neurogenesis in mesial temporal lobe epilepsy

Background

In mesial temporal lobe epilepsy, reduced hippocampal neurogenesis correlates with severe impairment in declarative learning and memory. The proinflammatory cytokines interleukin 1β and high-mobility group box 1 (HMGB1) and their receptors have key roles in generating and perpetuating seizures. This study aimed to assess their potential role in inhibiting neurogenesis in patients with mesial temporal lobe epilepsy and whether any effect would be pharmacologically reversible as a possible mechanism to restore learning and memory deficits.

Enalapril preserves sinus node function in a canine atrial fibrillation model induced by rapid atrial pacing

METHODS AND RESULTS: Seventeen beagles were pretreated with either placebo (group I, n = 9) or enalapril 1 mg/kg/day (group II, n = 8) and paced at 500/min from the right atrial appendage for 4 weeks. Every week, corrected sinus node recovery time (CSNRT) and sinus cycle length (SCL) were measured. Quantitative analysis of interstitial fibrosis (IF) and adipose tissue (AT) in the SN was performed with Masson's trichrome stain, and apoptosis of the sinus nodal cells were detected with terminal deoxynucleotidyl transferase nick end-labeling. In group I, rapid atrial pacing prolonged both CSNRT and SCL. After 4 weeks of pacing, CSNRT and SCL were significantly shorter in group II (CSNRT, 410 +/- 37 msec; SCL, 426 +/- 34 msec) than in group I (CSNRT, 717 +/- 52 msec, P < 0.005; SCL, 568 +/- 73 msec, P < 0.05). Both IF and AT of the SN were greater in group I (IF, 9.7 +/- 1.9%; AT, 32.6 +/- 5.9%) than in seven sham dogs (IF, 2.4 +/- 0.9%, P < 0.05; AT, 4.0 +/- 1.7%, P < 0.05) and in group II dogs (IF, 4.0 +/- 2.0%, P < 0.05; AT, 4.0 +/- 1.7%, P < 0.05). End-labeling assay was positive in three of nine dogs in group I, but negative in group II and sham dogs. CONCLUSIONS: Rapid atrial pacing impaired SN function through IF and AT of the SN. Enalapril prevented these pacing-induced degenerative changes and improved SN function.     

Nicorandil preserves the function of the mitochondrial phosphorylative and oxidative system in an animal model of global ischemia-reperfusion.

Ischemia followed by reperfusion (IR) negatively affects mitochondrial function. At the level of the oxidative-phosphorylative system, IR inhibits the respiratory complexes and ATP synthase, and increases the passive leak of protons through the inner mitochondrial membrane, uncoupling respiration from phosphorylation, decreasing mitochondrial potential and, consequently, ATP production. Drugs that minimize the mitochondrial damage induced by IR may prove to be clinically effective. In the present work, we analyzed the impact of nicorandil, a mitochondrial ATP-sensitive potassium channel agonist, on mitochondrial dysfunction at the level of the oxidative-phosphorylative system of rat hearts subjected to IR. The decrease in the respiratory control ratio (RCR) induced by IR leads to the conclusion that IR has a negative impact on the activity of the mitochondrial respiratory system, uncoupling oxidation from phosphorylation. This effect is reversed by nicorandil, which increases not only RCR, but also the ADP/O ratio. Regarding respiratory rate, state 3 rate was approximately the same for all the experimental groups, while state 4 rate was lower for the group where IR was induced in the presence of nicorandil. This result is in accordance with the data obtained for the RCR and ADP/O. State 4 rate is most affected by uncoupling, given that it is controlled by proton leak. Mitochondria subjected to IR in the presence of nicorandil have a lower state 4 rate, i.e. they are less uncoupled. From these results we conclude that nicorandil preserves the function of mitochondria subjected to IR in terms of both respiration and phosphorylative capacity.     

Positive end-expiratory pressure preserves surfactant function in preterm lambs.

Ventilation style influences lung injury and the amount of large-aggregate biophysically active surfactant in adult lungs. We asked how positive end-expiratory pressures (PEEP) would influence clinical responses and surfactant pools in surfactant-treated preterm lambs ventilated for 7 h with tidal volumes (VT) of 10 ml/kg. The 126-d gestation preterms were delivered and treated with 100 mg/kg recombinant human surfactant protein C (rSP-C) containing surfactant and ventilated with zero, 4, or 7 cm H(2)O of PEEP. A comparison group was treated with natural sheep surfactant and ventilated with zero PEEP. Physiologic measurements were similar for lambs treated with rSP-C surfactant and natural surfactant. PEEP 4 and 7 improved oxygenation and compliance relative to either group of lambs ventilated with PEEP zero. The maximal lung volumes measured at 40 cm H(2)O pressure after 7 h ventilation for the PEEP 4 and 7 groups were more than double those measured for either PEEP zero group. Alveolar surfactant pools were larger for the PEEP 7 group, and the large-aggregate fraction was increased for the PEEP 4 and 7 groups, resulting in large-aggregate pool sizes that were 3-fold higher for the PEEP 4 and 4-fold higher for the PEEP 7 groups relative to the PEEP zero group treated with rSP-C surfactant. All large-aggregate surfactants lowered minimal surface tensions of a captive bubble to less than 5 mN/m. In preterm surfactant-treated lambs PEEP improved lung function and maintained more of an rSP-C surfactant in the biophysically active form.     

Ethyl pyruvate enhances ATP levels, reduces oxidative stress and preserves cardiac function in a rat model of off-pump coronary bypass

BACKGROUND: Off-pump coronary artery bypass grafting is associated with transient periods of myocardial ischemia during revascularization resulting in myocardial contractile dysfunction and oxidative injury. The purpose of this study was to investigate the efficacy of ethyl pyruvate as a myocardial protective agent in a rat model of off-pump coronary artery bypass grafting associated with transient myocardial dysfunction without infarction. METHODS: Wistar rats were subjected to transient ischemia via 10 min occlusion of the LAD coronary artery followed by 10 min of reperfusion. Animals received an IV bolus of Ringer's solution as a control (n=10) or Ringer's ethyl pyruvate (n=10) immediately before the initiation of ischemia and reperfusion. Myocardial ATP and lipid peroxidation levels were quantified for an estimation of energetics and oxidative stress, respectively. In vivo cardiac function was assessed throughout the ischemia and reperfusion periods. RESULTS: Ethyl pyruvate significantly increased myocardial ATP levels compared to controls (2650+/-759 nmol/g versus 892+/-276 nmol/g, p=0.04). Myocardial oxidative stress was significantly reduced in animals treated with ethyl pyruvate compared to controls (70.4+/-2.6 nmol/g versus 81.8+/-2.4 nmol/g, p=0.04). dP/dt max and cardiac output were significantly greater in the ethyl pyruvate group compared to controls during ischemia and reperfusion. CONCLUSIONS: Ethyl pyruvate enhances myocardial ATP levels, reduces oxidative stress, and preserves myocardial function in a model of transient ischemia/reperfusion injury not subject to myocardial infarction.     

Variation in DISC1 affects hippocampal structure and function and increases risk for schizophrenia

Disrupted-in-schizophrenia 1 (DISC1) is a promising schizophrenia candidate gene expressed predominantly within the hippocampus. We typed 12 single-nucleotide polymorphisms (SNPs) that covered the DISC1 gene. A three-SNP haplotype [hCV219779 (C)-rs821597 (G)-rs821616 (A)] spanning 83 kb of the gene was associated with schizophrenia in a family-based sample (P = 0.002). A common nonconservative SNP (Ser704Cys) (rs821616) within this haplotype was associated with schizophrenia (P = 0.004). Based on primary expression of DISC1 in hippocampus, we hypothesized that allelic variation at Ser704Cys would have a measurable impact on hippocampal structure and function as assayed via specific hippocampus-related intermediate phenotypes. In addition to overtransmission in schizophrenia, the Ser allele was associated with altered hippocampal structure and function in healthy subjects, including reduced hippocampal gray matter volume and altered engagement of the hippocampus during several cognitive tasks assayed with functional magnetic resonance imaging. These convergent data suggest that allelic variation within DISC1, either at Ser704Cys or haplotypes monitored by it, increases the risk for schizophrenia and that the mechanism of this effect involves structural and functional alterations in the hippocampal formation.     

Spatio-temporal heterogeneity in hippocampal metabolism in control and epilepsy conditions

The hippocampus’s dorsal and ventral parts are involved in different operative circuits, the functions of which vary in time during the night and day cycle. These functions are altered in epilepsy. Since energy production is tailored to function, we hypothesized that energy production would be space- and time-dependent in the hippocampus and that such an organizing principle would be modified in epilepsy. Using metabolic imaging and metabolite sensing ex vivo, we show that the ventral hippocampus favors aerobic glycolysis over oxidative phosphorylation as compared to the dorsal part in the morning in control mice. In the afternoon, aerobic glycolysis is decreased and oxidative phosphorylation increased. In the dorsal hippocampus, the metabolic activity varies less between these two times but is weaker than in the ventral. Thus, the energy metabolism is different along the dorsoventral axis and changes as a function of time in control mice. In an experimental model of epilepsy, we find a large alteration of such spatiotemporal organization. In addition to a general hypometabolic state, the dorsoventral difference disappears in the morning, when seizure probability is low. In the afternoon, when seizure probability is high, the aerobic glycolysis is enhanced in both parts, the increase being stronger in the ventral area. We suggest that energy metabolism is tailored to the functions performed by brain networks, which vary over time. In pathological conditions, the alterations of these general rules may contribute to network dysfunctions.

Energy production in brain cells is assumed to be optimized to perform tasks or activities in a brain region–specific manner (1–3). Simultaneously, neuronal activity should be adapted to minimize the energy expenditure required for its fueling (4, 5). Several metabolic pathways are available for energy production, in particular glycolysis and oxidative phosphorylation (1). Whether specific metabolic pathways are preferably recruited in different brain areas in a task-dependent manner is not known. The hippocampus is an ideal region to test this hypothesis. The dorsal hippocampus (DH) is involved in learning and memory associated with navigation, exploration, and locomotion, whereas the ventral hippocampus (VH) is involved in motivational and emotional behavior (6–8). These functions are supported by very distinct anatomical (9, 10), morphological (11–13), molecular (14–19), and electrophysiological (12, 13, 20–23) properties of hippocampal cells. The hippocampus structure is also highly heterogeneous at the gene level, from its dorsal to its ventral tip (24, 25), which may serve as a substrate for different functional networks related to cognition and emotion to emerge (7, 26, 27). Given the hippocampus’s heterogeneity, from structure to function, along its dorsoventral axis, our first hypothesis is that energy production is different between the VH and the DH.If energy production is tailored to a given structure–function relationship, we predicted that a change in energy production should accompany a change in the hippocampus’s functional state. Epilepsy is a particularly relevant situation to test this hypothesis. The different types of epilepsies are associated with numerous metabolic and bioenergetic alterations (28). Hypometabolism of epileptic regions is a common signature of mesial temporal lobe epilepsy (TLE) in humans and animal models, such as the pilocarpine model (29, 30). Importantly, in patients with TLE, only the temporal part (including the hippocampus) is epileptogenic. The temporal part corresponds to the ventral part in rodents, which has been identified as the epileptogenic region in the pilocarpine mode (31). Our second hypothesis is that any dorsoventral organization of energy metabolism found in control condition is altered in epilepsy.Finally, hippocampal functions demonstrate circadian regulation (32), in particular place cell properties (33) and long-term synaptic plasticity (34) as well as memory and learning processes (35, 36). Our third hypothesis is that energy production, specifically the respective contributions of oxidative phosphorylation and aerobic glycolysis (3), vary as a function of the time of the day in control and epilepsy.To test these three hypotheses, we used an ex vivo approach to evoke an energy-demanding electrophysiological activity standardized for the three independent variables considered here: time, region (DH/VH), and perturbation (control/epilepsy). As a first step toward a better understanding of the time regulation of hippocampal metabolism, we considered two time points during the night/day cycle: Zeitgeber 3 (ZT3) and Zeitgeber 8 (ZT8), as they correspond to low and high seizure probability in the TLE model used (37). We found that the control DH and VH have distinct time-dependent metabolic signatures regarding glycolysis and oxidative phosphorylation. In experimental epilepsy, there is no more dissociation between DH and VH at ZT3, but the regional difference reappears at ZT8.