Marie-Aline Laute

Effects of anti–glutamic acid decarboxylase antibodies associated with neurological diseases

Glutamic acid decarboxylase (GAD) catalyzes the conversion of glutamic acid into GABA. GAD autoantibodies (GAD‐Ab) have been described in diabetes mellitus and in diseases involving the central nervous system such as stiff‐person syndrome and cerebellar ataxia. However, the pathogenic role of GAD‐Ab in neurological diseases remains a matter of debate.

Sleep deprivation decreases phase-shift responses of circadian rhythms to light in the mouse: role of serotonergic and metabolic signals

The circadian pacemaker in the suprachiasmatic nuclei is primarily synchronized to the daily light-dark cycle. The phase-shifting and synchronizing effects of light can be modulated by non-photic factors, such as behavioral, metabolic or serotonergic cues. The present experiments examine the effects of sleep deprivation on the response of the circadian pacemaker to light and test the possible involvement of serotonergic and/or metabolic cues in mediating the effects of sleep deprivation. Photic phase-shifting of the locomotor activity rhythm was analyzed in mice transferred from a light-dark cycle to constant darkness, and sleep-deprived for 8 h from Zeitgeber Time 6 to Zeitgeber Time 14. Phase-delays in response to a 10-min light pulse at Zeitgeber Time 14 were reduced by 30% in sleep-deprived mice compared to control mice, while sleep deprivation without light exposure induced no significant phase-shifts. Stimulation of serotonin neurotransmission by fluoxetine (10 mg/kg), a serotonin reuptake inhibitor that decreases light-induced phase-delays in non-deprived mice, did not further reduce light-induced phase-delays in sleep-deprived mice. Impairment of serotonin neurotransmission with p-chloroamphetamine (three injections of 10 mg/kg), which did not increase light-induced phase-delays in non-deprived mice significantly, partially normalized light-induced phase-delays in sleep-deprived mice. Injections of glucose increased light-induced phase-delays in control and sleep-deprived mice. Chemical damage of the ventromedial hypothalamus by gold-thioglucose (600 mg/kg) prevented the reduction of light-induced phase-delays in sleep-deprived mice, without altering phase-delays in control mice. Taken together, the present results indicate that sleep deprivation can reduce the light-induced phase-shifts of the mouse suprachiasmatic pacemaker, due to serotonergic and metabolic changes associated with the loss of sleep.

A possible mechanism for the beneficial effect of ethanol in essential tremor

Background:  Essential tremor is one of the most common movement disorders in elderly people. The hypothesis of a disregulation of N‐methyl‐d‐aspartate (NMDA) pathways has been suggested. It was shown experimentally that infusion of NMDA in cerebellar nuclei down‐regulates glutamate release.

The cerebellum modulates rodent cortical motor output after repetitive somatosensory stimulation

OBJECTIVE:To analyze the possible role of the cerebellum in the modulation of cortical motor output associated with repetitive electrical stimulation of the sciatic nerve in the rat. METHODS:A sustained somatosensory stimulation induces an increase in the intensity of the response of the rodent motor cortex. Wistar rats were anesthetized for surgical preparation using a continuous infusion of chloral hydrate. We analyzed the response evoked by electrical stimulation of the right motor cortex before (basal condition) and after peripheral electrical stimulation of the left sciatic nerve in rats with no cerebellar intervention (n = 6), and in control rats with Ringer’s infusion via a microdialysis probe (n = 8) implanted in the left cerebellar nuclei. In addition, we investigated the effects of 1) the administration of ethanol (20 mmol/L) in the left cerebellar nuclei (n = 5); 2) the administration of tetrodotoxin (10 &mgr;mol/L), a sodium channel blocker, in the left cerebellar nuclei (n = 5); 3) electrical stimulation by deep cerebellar stimulation (frequency 100 Hz) on the left side (n = 5); or 4) electrical stimulation of the cerebellar nuclei on the right side (100 Hz; n = 6). For peripheral stimulation, all of the animals received 1 hour of electrical stimulation. Trains of stimulation consisted of five stimuli (duration of 1 stimulus, 1 ms) at a rate of 10 Hz. During stimulation of the motor cortex, peak-to-peak amplitudes in responses of the left calf muscle were analyzed. Motor threshold was defined as the lowest intensity eliciting at least 5 of 10 evoked responses with an amplitude greater than 20 &mgr;V. The intensity used was 130% of the motor threshold. RESULTS:In the basal condition (before repetitive stimulation), amplitudes of motor responses were similar in the six groups of rats (P = 0.40). In rats without cerebellar intervention, peripheral electrical stimulation was associated with an increase of motor response to 147.4 ± 8.5% of baseline (P < 0.001). In rats with Ringer’s infusion, the motor response increased to 141.6 ± 7.9% of baseline (P < 0.001). The administration of ethanol in the cerebellum prevented the enhancement of the response ipsilaterally. The mean ± standard deviation (SD) of motor responses was 105.7 ± 6.2% of baseline measurements after stimulation of the sciatic nerve (P = 0.36). The same observation was made after the infusion of tetrodotoxin (mean ± SD of motor responses: 107.1 ± 7.4% after peripheral stimulation [P = 0.19] and after electrical stimulation of the cerebellum on the left side [mean ± SD of motor responses, 104.3 ± 8.5% after peripheral stimulation, P = 0.40]). However, electrical stimulation of cerebellar nuclei on the right side did not impair the modulation of cortical motor output by sciatic nerve stimulation (mean ± SD of motor responses, 148.4 ± 5.8% after peripheral stimulation, P < 0.001). CONCLUSION:Until now, the increase of motor output after peripheral nerve stimulation has been considered as a plasticity directly and solely dependent on cortical structures. We demonstrate that the cerebellum plays a key role in this form of neural plasticity.

Depression of extra-cellular GABA and increase of NMDA-induced nitric oxide following acute intra-nuclear administration of alcohol in the cerebellar nuclei of the rat

Gamma-aminobutyric acid (GABA) and nitric oxide are two key-transmitters in cerebellar nuclei, the major output of cerebellar circuitry. The aims of this study were to investigate the effects of acute intra-cerebellar administration of ethanol (20 mM) on extra-cellular levels of GABA and on the NMDA-induced nitric oxide (NO) production using microdialysis in the rat. We also studied: (i) the effects of a pre-administration of DNQX, a specific antagonist of AMPA receptors, on NO production, (ii) the effects of a pre-administration of 7-NI (7-nitroindazole, an inhibitor of neuronal nitric oxide synthase NOS) and APV (D-2-amino-5-phosphonovaleric acid, a specific blocker of the NMDA type glutamate receptors) on the actions of alcohol/NMDA on glutamate receptors, and (iii) thein vivo interaction between DNQX, ethanol and NMDA receptor activation. We found that ethanol decreased the amount of extra-cellular GABA, and that this effect was counterbalanced by administration of tiagabine 1 mg/kg, a potent inhibitor of GAT-1 GABA transporter, given by the i.p. route. In loco administration of NMDA increased the levels of NO, as previously reported. A pre-administration of DNQX (500 microM) increased significantly the production of NO up to toxic levels, as well as ethanol administration. A preadministration of 7-NI or APV reduced significantly the amounts of NO when NMDA and alcohol were infused simultaneously. The combination of ethanol with DNQX was associated with a marked enhancement of the concentrations of NO. The activity of GAT-1 in cerebellar nuclei and around this target, including in glial cells expressing GAT-1 activated by ambient GABA, seems to be spared by ethanol. Tiagabine could be considered as a candidate for future investigational treatments of acute ethanol-induced dysfunction of cerebellar nuclei. We found a potentiation of the production of NO when AMPA antagonists are given simultaneously to ethanol. The hypothesis of AMPA neurotoxicity, which has convincing arguments during chronic exposure, is challenged in this model of acute cerebellar nuclear toxicity of alcohol.

Interferon regulatory factor 3 deficiency leads to interleukin-17-mediated liver ischemia-reperfusion injury.

Interferon regulatory factor 3 (IRF3) is an important transcription factor in Toll‐like receptor 4 (TLR4) signaling, a pathway that is known to play a critical role in liver ischemia‐reperfusion injury. In order to decipher the involvement of IRF3 in this setting, we first compared the intensity of hepatic lesions in IRF3‐deficient versus wildtype mice. We found increased levels of blood transaminases, enhanced liver necrosis, and more pronounced neutrophil infiltrates in IRF3‐deficient mice. Neutrophil depletion by administration of anti‐Ly6G monoclonal antibody indicated that neutrophils play a dominant role in the development of severe liver necrosis in IRF3‐deficient mice. Quantification of cytokine genes expression revealed increased liver expression of interleukin (IL)‐12/IL‐23p40, IL‐23p19 messenger RNA (mRNA), and IL‐17A mRNA in IRF3‐deficient versus wildtype (WT) mice, whereas IL‐27p28 mRNA expression was diminished in the absence of IRF3. The increased IL‐17 production in IRF3‐deficient mice was functionally relevant, as IL‐17 neutralization prevented the enhanced hepatocellular damages and liver inflammation in these animals. Evidence for enhanced production of IL‐23 and decreased accumulation of IL‐27 cytokine in M1 type macrophage from IRF3‐deficient mice was also observed after treatment with lipopolysaccharide, a setting in which liver gamma‐delta T cells and invariant natural killer T cells were found to be involved in IL‐17A hyperproduction. Conclusion: IRF3‐dependent events downstream of TLR4 control the IL‐23/IL‐17 axis in the liver and this regulatory role of IRF3 is relevant to liver ischemia‐reperfusion injury. (HEPATOLOGY 2013)

Effect of pergolide on endogenous and exogenous L-DOPA metabolism in the rat striatum: a microdialysis study.

We used a model of intrastriatal microdialysis in freely moving rats to study the effect of pergolide, a mixed D1/D2 dopamine (DA) receptor agonist with predominant D2 action in vivo, on the biotransformation of endogenous and exogenous L-DOPA. Levels of L-DOPA, DA, DOPAC, HVA and 5-HIAA were measured by high performance liquid chromatography. Pergolide (50 μg/kg, i.p.) caused a 47%, 65% and 70% decrease in basal striatal extracellular (EC) levels of DOPAC, HVA and DA, respectively. L-DOPA (100 mg/kg, i.p.), injected 2 hours after carbidopa, produced significant increase in EC levels of L-DOPA, DOPAC, HVA and DA in rats with and without local perfusion of 10-4 M pergolide. The DOPAC peak value was lower and was reached 60 minutes later in the group with pergolide. This study demonstrated inhibitory effects of pergolide on endogenous DA release and influence of pergolide on exogenous L-DOPA biotransformation.

Cerebellar spongiform degeneration induced by acute lithium intoxication in the rat.

Cerebellar syndrome has been described after acute lithium intoxication in human. Neuropathological studies have demonstrated neuronal loss and spongiosis in the cerebellum. We describe an animal model of acute lithium-induced cerebellar degeneration. Five hours following administration of lithium chloride (250 mg/kg, i.p.), the cerebellar white matter of seven rats out 14 exhibited extensive spongiform changes. Microdialysis study in the rat cerebellar cortex demonstrated basal concentrations of dopamine (DA), hydroxy-3-methoxyphenylacetic acid (HVA) and 5-hydroxy-3-indolacetic acid (5-HIAA). These metabolites were unaffected by acute lithium intoxication suggesting that the cerebellar toxicity is not due to a modification of dopaminergic or serotoninergic neurotransmission.

Interaction between repetitive stimulation of the sciatic nerve and functional ablation of cerebellar nucleus interpositus in the rat

It is established that cerebellar nuclei exert a significant effect on the excitability of spinal neurons. However, their output is heterogeneous. Conditioning trains of dentate nucleus stimuli are known to modify the post-synaptic potentials evoked in motoneurons by stimulation of group Ia and Ib afferents in appropriate peripheral nerves. The role of the interpositus nucleus in the modulation of the excitability of rat spinal cord remains unclear. We investigated the interactions between tetrodotoxin (TTX)-induced inactivation of the interpositus cerebellar nuclei and repetitive electrical stimulation of the ipsilateral sciatic nerve (proximal segment) in the anesthetized rat. TTX (10 microM) was administered in cerebellar nuclei by the technique of microdialysis (coordinates of the extremity of the guide related to bregma: AP: -11.6, L: +2.3, V: -4.6). Peripheral stimulation consisted of trains of electric stimuli at a rate of 10 Hz, which were repeated every second during 1 hour. Stimulus intensity was adjusted to produce constant somatosensory evoked potentials. H-reflex, F-wave and M responses of the plantaris muscles were analysed ipsilaterally. H-reflex recruitment curve, Hmax/Mmax ratios, F-wave persistence and mean F/mean M ratios were studied. Functional blockade of cerebellar interpositus nucleus reduced the slope of H-reflex recruitment curve without affecting the Hmax/Mmax ratio, and depressed both F-waves persistence and mean F/mean M ratios. Concomitant repetitive stimulation of the sciatic nerve counteracted the depression of the H-reflex recruitment curve, without interacting with F-waves depression. Our results (1) show that TTX-sensitive sodium channels in cerebellar nucleus interpositus modulate the H-reflex recruitment, and (2) reveal an interaction between TTX-sensitive sodium channels in cerebellar nuclei and afferent repetitive activity not described so far.

Effects of levetiracetam on the production of nitric oxide--an in vivo study.

Sirs: Nitric oxide (NO) is a diffusible gas playing a critical role in signal transduction in the central nervous system, with major functions in neuronal signalling and plasticity [13]. It has unique properties as it can diffuse through cell membranes without using transporters. NO is necessary for neuron survival and it attenuates neurotoxicity mediated by voltage-gated calcium channels [6, 12]. However, in the absence of trophic factors NO can become toxic by forming peroxynitrite, a toxin formed by the diffusion-limited reaction between NO and superoxide anions [2, 3]. The cerebellum is the area of the brain with the highest nitric oxide synthase (NOS) activity, which is triggered by activation of glutamate receptors [14]. So far, most studies of the role of NO in the cerebellum have focused on the cerebellar cortex, despite the major contribution of deep cerebellar nuclei in shaping the output signals coming from the cerebellar circuitry. Neurons in the deep nuclei receive glutamatergic inputs from branches of climbing and mossy fibers via both NMDA and the AMPA receptors [1, 4]. Levetiracetam is a new antiepileptic drug with a high level of binding in 4 regions of the brain (the dentate gyrus, the superior colliculus, thalamic nuclei, and the cerebellum) and which is characterized by a unique activity profile [7]. It is currently used both as an adjunctive treatment and as a monotherapy in partial seizures. In cultured astrocytes, it was recently shown that levetiracetam increases the expression of inducible NOS (iNOS) and also of BDNF [2]. Levetiracetam stimulates the expression of iNOS in a concentration-dependent manner. Starting at about 0.3 mmol, NO synthesis can be effectively stimulated by NMDA receptors, but it remains to be determined whether levetiracetam effect on NO is due to an interaction with these receptors. In this study, we analyzed the in vivo interaction of levetiracetam with NMDA and AMPA pathways. The effects of levetiracetam on the production of NO were investigated using intra-cerebellar microdialysis in rats. In addition, we tested the hypothesis that DNQX, an AMPA antagonist, increases the production of NO following a pretreatment with levetiracetam. The experiments were approved by the Animal Care Committee of the Free University of Brussels (ULB). Microdialysis was performed in male Wistar rats (Charles River Laboratories; weight between 250 and 300 g). All the animals underwent cerebellar surgery on the left side, with surgical preparation using a loading dose of chloral hydrate ip (400 mg/kg). A microdialysis probe (CMA 12, CMA, Sweden) was inserted into the left cerebellar nuclei (AP: –11.6 mm, L: +2.3 mm, D: –5 mm; coordinates according to the Paxinos-Watson atlas [10]). The guide was fixed on the skull with dental cement. Rats were allowed 24 recovery time after the implantation of the guide cannula, and were given free access to food and water after surgery. Location of the probe was verified histologically at the end of the experiments after decapitation and removal of the cerebellum. NO production was measured using a two step assay method (colorimetric determination – Griess reaction, Active Motif, Belgium). The absorbance was read on a spectrophotometer at 540 nm with a reference wavelength of 620 nm. Linearity curves were analyzed for each rat. Perfusion of the probe was performed at a rate of 2 μl/ min (CMA-100 pump; dialysates collected every twenty minutes; volumes of 40 μl). To assess the reliability of the method and the stability of NO measurements, we analyzed the effects of Ringer infusion on NO measurements in 3 rats. Five successive microdialysates were collected for each rat (Fig. 1). At time 0, +20 min, +40 min, +60 min and +80 min, the mean value changed from 100 % to 109 %, 107 %, 108 %, 108 % respectively (ANOVA: p = 0.34). To validate our technique, we have also administered L-NAME (N-nitro-Larginine methyl ester), a NOS inhibitor, at a dose of 10 mg/kp by the ip route in 3 rats. This dose is one tenth of the dose used by Yamada and Nabeshima (100 mg/ kg) [14]. These authors observed a reduction of 43.9 ± 1.6 % of the basal levels after the administration of L-NAME (see Table 1 page 98). We found a mean reduction from 100 % to 88.1 % (Fig. 2), which is the range expected with the dose used in our study. In a first study (n = 8 control rats), we analyzed the effects of NMDA (10 mmol; administration during 80 minutes) on NO production. Basal levels were measured by averaging 3 consecutive stable dialysates samples [14]. In a second study, the protocol was composed of 3 steps: (1) preadministration of levetiracetam (2 doses: 3.6 μg and 36 μg, correLETTER TO THE EDITORS