Marcelo Duzzioni

GABA-A receptor modulators alter emotionality and hippocampal theta rhythm in an animal model of long-lasting anxiety

The cholinergic system is implicated in emotional regulation. The injection of non-convulsant doses of the muscarinic receptor agonist pilocarpine (PILO) induces long-lasting anxiogenic responses in rats evaluated at different time-points (24h to 3 months). To investigate the underlying mechanisms, rats treated with PILO (150mg/kg) were injected 24h or 1 month later with an anxiolytic (diazepam, 1mg/kg, DZP) or anxiogenic (pentylenetetrazole, 15mg/kg, PTZ) drug and evaluated in the elevated plus-maze (EPM). Prefrontal cortex (PFC) and hippocampal (HIP) electroencephalographic recordings and acetylcolinesterase (AChE) activity were also analyzed after PILO treatment. Anxiogenic responses observed in the EPM 24h or 1 month after PILO treatment (e.g., decreased time spent and number of entries into the open arms of the maze) were blocked by DZP but not affected by PTZ. No epileptiform events were registered in the HIP or PFC at 24h or 1 month after PILO injection, but enhanced theta activity was observed in the HIP. DZP decreased hippocampal theta of PILO-treated rats in contrast with PTZ, which increased this parameter in saline- and PILO-treated rats. The HIP and PFC AChE activity did not change after PILO treatment. Our findings demonstrate that the long-term effects on the emotionality of rats induced by PILO are associated with electrophysiological changes in the HIP and sensitive to pharmacological manipulation of the GABAergic system. The present work may support this new research model of long-lasting anxiety, while also highlighting the muscarinic system as a potential target involved in anxiety disorders.

NK1 receptors antagonism of dorsal hippocampus counteract the anxiogenic-like effects induced by pilocarpine in non-convulsive Wistar rats.

Recent evidence supports a role for the substance P (SP) in the control of anxiety and epilepsy disorders. Aversive stimuli alter SP levels and SP immunoreactivity in limbic regions, suggesting that changes in SP-NK1 receptor signaling may modulate the neuronal excitability involved in seizures and anxiogenesis. The involvement of NK1 receptors of the dorsal hippocampus and lateral septum in the anxiogenic-like effects induced by a single injection of pilocarpine (PILO) was examined in non-convulsive rats evaluated in the elevated plus-maze (EPM). Male Wistar rats were systemically injected with methyl-scopolamine (1mg/kg) followed 30 min later by saline or PILO (350 mg/kg) and only rats that did not present status epilepticus were used. One month later, vehicle or FK888 (100 pmol) - an NK1 receptor antagonist - were infused in the dorsal hippocampus or the lateral septum of the rats and then behaviorally evaluated in the EPM. Previous treatment with PILO decreased the time spent in and the frequency of entries in the open arms of the EPM, besides altering risk-assessment behaviors such as the number of unprotected head-dipping, protected stretch-attend postures and the frequency of open-arms end activity, showing thus a long-lasting anxiogenic-like profile. FK888 did not show any effect per se but inhibited the anxiogenic responses induced by PILO when injected into the dorsal hippocampus, but not into the lateral septum. Our data suggest that SP-NK1 receptor signaling of the dorsal hippocampus is involved in the anxiogenic-like profile induced by PILO in rats evaluated in the EPM test.

Genetic susceptibility in Juvenile Myoclonic Epilepsy: Systematic review of genetic association studies

Background Several genetic association investigations have been performed over the last three decades to identify variants underlying Juvenile Myoclonic Epilepsy (JME). Here, we evaluate the accumulating findings and provide an updated perspective of these studies. Methodology A systematic literature search was conducted using the PubMed, Embase, Scopus, Lilacs, epiGAD, Google Scholar and Sigle up to February 12, 2016. The quality of the included studies was assessed by a score and classified as low and high quality. Beyond outcome measures, information was extracted on the setting for each study, characteristics of population samples and polymorphisms. Results Fifty studies met eligibility criteria and were used for data extraction. With a single exception, all studies used a candidate gene approach, providing data on 229 polymorphisms in or near 55 different genes. Of variants investigating in independent data sets, only rs2029461 SNP in GRM4, rs3743123 in CX36 and rs3918149 in BRD2 showed a significant association with JME in at least two different background populations. The lack of consistent associations might be due to variations in experimental design and/or limitations of the approach. Conclusions Thus, despite intense research evidence established, specific genetic variants in JME susceptibility remain inconclusive. We discussed several issues that may compromise the quality of the results, including methodological bias, endophenotype and potential involvement of epigenetic factors. PROSPERO registration number CRD42016036063

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

Neuropathological studies often use autopsy brain tissue as controls to evaluate changes in protein or RNA levels in several diseases. In mesial temporal lobe epilepsy (MTLE), several genes are up or down regulated throughout the epileptogenic and chronic stages of the disease. Given that postmortem changes in several gene transcripts could impact the detection of changes in case-control studies, we evaluated the effect of using autopsy specimens with different postmortem intervals (PMI) on differential gene expression of the Pilocarpine (PILO)induced Status Epilepticus (SE) of MTLE. For this, we selected six genes (Gfap, Ppia, Gad65, Gad67, Npy, and Tnf-α) whose expression patterns in the hippocampus of PILO-injected rats are well known. Initially, we compared hippocampal expression of naïve rats whose hippocampi were harvested immediately after death (0h-PMI) with those harvested at 6h postmortem interval (6h-PMI): Npy and Ppia transcripts increased and Tnf-α transcripts decreased in the 6h-PMI group (p<0.05). We then investigated if these PMI-related changes in gene expression have the potential to adulterate or mask RT-qPCR results obtained with PILO-injected rats euthanized at acute or chronic phases. In the acute group, Npy transcript was significantly higher when compared with 0h-PMI rats, whereas Ppia transcript was lower than 6h-PMI group. When we used epileptic rats (chronic group), the RT-qPCR results showed higher Tnf-α only when compared to 6h-PMI group. In conclusion, our study demonstrates that PMI influences gene transcription and can mask changes in gene transcription seen during epileptogenesis in the PILO-SE model. Thus, to avoid erroneous conclusions, we strongly recommend that researchers account for changes in postmortem gene expression in their experimental design.

Identification of microRNAs with Dysregulated Expression in Status Epilepticus Induced Epileptogenesis

The involvement of miRNA in mesial temporal lobe epilepsy (MTLE) pathogenesis has increasingly become a focus of epigenetic studies. Despite advances, the number of known miRNAs with a consistent expression response during epileptogenesis is still small. Addressing this situation requires additional miRNA profiling studies coupled to detailed individual expression analyses. Here, we perform a miRNA microarray analysis of the hippocampus of Wistar rats 24 hours after intra-hippocampal pilocarpine-induced Status Epilepticus (H-PILO SE). We identified 73 miRNAs that undergo significant changes, of which 36 were up-regulated and 37 were down-regulated. To validate, we selected 5 of these (10a-5p, 128a-3p, 196b-5p, 352 and 324-3p) for RT-qPCR analysis. Our results confirmed that miR-352 and 196b-5p levels were significantly higher and miR-128a-3p levels were significantly lower in the hippocampus of H-PILO SE rats. We also evaluated whether the 3 miRNAs show a dysregulated hippocampal expression at three time periods (0h, 24h and chronic phase) after systemic pilocarpine-induced status epilepticus (S-PILO SE). We demonstrate that miR-128a-3p transcripts are significantly reduced at all time points compared to the naïve group. Moreover, miR-196b-5p was significantly higher only at 24h post-SE, while miR-352 transcripts were significantly up-regulated after 24h and in chronic phase (epileptic) rats. Finally, when we compared hippocampi of epileptic and non-epileptic humans, we observed that transcript levels of miRNAs show similar trends to the animal models. In summary, we successfully identified two novel dysregulated miRNAs (196b-5p and 352) and confirmed miR-128a-3p downregulation in SE-induced epileptogenesis. Further functional assays are required to understand the role of these miRNAs in MTLE pathogenesis.

Evidence for involvement of NK3 receptors in the anxiogenic-like effect of SP6-11(C-terminal), a metabolite of substance P, in rats evaluated in the elevated plus-maze

INTRODUCTIONnSubstance P (SP) is a neuropeptide widely expressed throughout the fear-processing pathways of the brain. SP is cleaved by several proteolytic enzymes in amino (N-) and carboxy (C-) terminal sequences, which can have biological activities per se. We have previously shown that the anxiogenic-like effects elicited by SP6-11(C-terminal), a specific metabolite of SP, are mediated via NK1 and NK2 receptors. Nevertheless, there are evidences that C-terminal fragments may have a greater affinity for NK3 receptors.nnnOBJECTIVESnThe aim of the present study was to further investigate the possible involvement of NK3 receptors in the anxiogenic-like effects induced by SP6-11(C-terminal).nnnMETHODSnAdult male Wistar rats were intracerebroventricularly (i.c.v.) treated with SR142801 (NK3 receptors antagonist) or vehicle one minute to prior SP6-11(C-terminal) or vehicle. Other experimental groups received SP6-11(C-terminal) or vehicle i.c.v. one minute prior to senktide (NK3 receptors agonist) or vehicle. After five minutes, the animals were behaviorally evaluated in the elevated plus-maze test (EPM).nnnRESULTSnSR142801 (100 pmol) or SP6-11(C-terminal) (10 pmol) reduced all the parameters of open-arms exploration and increased the number of protected stretch-attend postures in the EPM, indicating an anxiogenic-like effect. Senktide (10 pmol) promoted an opposite effect on these behavioral parameters, characterizing an anxiolytic-like profile. Pretreatment with SR142801, in an ineffective dose, potentiated the SP6-11-induced anxiety, especially in the unprotected head-dipping and protected stretch-attend postures behaviors. Moreover, the anxiolytic-like effect induced by senktide (1 pmol) was prevented by SP6-11.nnnCONCLUSIONSnOur results give support to the involvement of NK3 receptors in the anxiogenic-like actions of SP6-11(C-terminal), where this metabolite seems to behave as an antagonist, in a way similar to SR142801.

Inhibition of sodium glucose cotransporters following status epilepticus induced by intrahippocampal pilocarpine affects neurodegeneration process in hippocampus.

Temporal lobe epilepsy (TLE) is characterized by spontaneous recurrent seizures, starting from secondary functional disorders due to several insults, including self-sustaining continuous seizures identified as status epilepticus (SE). Although hypoglycemia has been associated with SE, the effect of inhibition of the Na(+)/glucose cotransporters (SGLTs) on hippocampus during SE is still unknown. Here we evaluated the functional role of SGLT in the pattern of limbic seizures and neurodegeneration process after pilocarpine (PILO)-induced SE. Vehicle (VEH, 1μL) or phlorizin, a specific SGLT inhibitor (PZN, 1μL, 50μg/μL), was administered in the hippocampus of rats 30min before PILO (VEH+PILO or PZN+PILO, respectively). The limbic seizures were classified using the Racines scale, and the amount of wet dog shakes (WDS) was quantified before and during SE. Neurodegeneration process was evaluated by Fluoro-Jade C (FJ-C), and FJ-C-positive neurons (FJ-C+) were counted 24h and 15days after SE. The PZN-treated rats showed higher (p<0.05) number of WDS when compared with VEH+PILO. There was no difference in seizure severity between PZN+PILO and VEH+PILO groups. However, the pattern of limbic seizures significantly changed in PZN+PILO. Indeed, the class 5 seizures repeated themselves more times (p<0.05) than the other classes in the PZN group at 50min after SE induction. The PZN+PILO animals had a higher (p<0.05) number of FJ-C+ cells in the dentate gyrus (DG), hilus, and CA3 and CA1 of hippocampus, when compared with VEH+PILO. The PZN+PILO animals had a decreased number (p<0.05) of FJ-C+ cells in CA1 compared with VEH+PILO 15days after SE induction. Taken together, our data suggest that SGLT inhibition with PZN increased the severity of limbic seizures during SE and increased neurodegeneration in hippocampus 24h after SE, suggesting that SGLT1 and SGLT2 could participate in the modulation of earlier stages of epileptogenic processes.

Maternal, fetal and neonatal consequences associated with the use of crack cocaine during the gestational period: a systematic review and meta-analysis

ObjectiveCrack cocaine consumption is one of the main public health challenges with a growing number of children intoxicated by crack cocaine during the gestational period. The primary goal is to evaluate the accumulating findings and to provide an updated perspective on this field of research.MethodsMeta-analyses were performed using the random effects model, odds ratio (OR) for categorical variables and mean difference for continuous variables. Statistical heterogeneity was assessed using the I-squared statistic and risk of bias was assessed using the Newcastle–Ottawa Quality Assessment Scale. Ten studies met eligibility criteria and were used for data extraction.ResultsThe crack cocaine use during pregnancy was associated with significantly higher odds of preterm delivery [odds ratio (OR), 2.22; 95% confidence interval (CI), 1.59–3.10], placental displacement (OR, 2.03; 95% CI 1.66–2.48), reduced head circumference (−u20091.65xa0cm; 95% CI −u20093.12 to −u20090.19), small for gestational age (SGA) (OR, 4.00; 95% CI 1.74–9.18) and low birth weight (LBW) (OR, 2.80; 95% CI 2.39–3.27).ConclusionThis analysis provides clear evidence that crack cocaine contributes to adverse perinatal outcomes. The exposure of maternal or prenatal crack cocaine is pointedly linked to LBW, preterm delivery, placental displacement and smaller head circumference.

Psychopharmacological effects and safety of styryl-2-pyrones and dihydrostyryl-2-pyrones-rich fraction from Polygala sabulosa: absence of withdrawal syndrome and tolerance to anxiolytic-like and anticonvulsant effects

To investigate whether mice develop tolerance to the anxiolytic‐like and anticonvulsant effects of subchronic treatment with EA (the styryl‐2‐pyrones and dihydrostyryl‐2‐pyrones‐rich fraction of Polygala sabulosa), as well as any withdrawal symptoms after abrupt discontinuation; to compare the effects of EA with those of diazepam (DZP) on withdrawal‐induced anxiety; and to evaluate the toxicity of EA according to OECD guidelines.

Rhythms of Core Clock Genes and Spontaneous Locomotor Activity in Post-Status Epilepticus Model of Mesial Temporal Lobe Epilepsy

The interaction of Mesial Temporal Lobe Epilepsy (mTLE) with the circadian system control is apparent from an oscillatory pattern of limbic seizures, daytimes effect on seizure onset and the efficacy of antiepileptic drugs. Moreover, seizures per se can interfere with the biological rhythm output, including circadian oscillation of body temperature, locomotor activity, EEG pattern as well as the transcriptome. However, the molecular mechanisms underlying this cross-talk remain unclear. In this study, we systematically evaluated the temporal expression of seven core circadian transcripts (Bmal1, Clock, Cry1, Cry2, Per1, Per2, and Per3) and the spontaneous locomotor activity (SLA) in post-status epilepticus (SE) model of mTLE. Twenty-four hour oscillating SLA remained intact in post-SE groups although the circadian phase and the amount and intensity of activity were changed in early post-SE and epileptic phases. The acrophase of the SLA rhythm was delayed during epileptogenesis, a fragmented 24 h rhythmicity and extended active phase length appeared in the epileptic phase. The temporal expression of circadian transcripts Bmal1, Cry1, Cry2, Per1, Per2, and Per3 was also substantially altered. The oscillatory expression of Bmal1 was maintained in rats imperiled to SE, but with lower amplitude (A = 0.2) and an advanced acrophase in the epileptic phase. The diurnal rhythm of Cry1 and Cry2 was absent in the early post-SE but was recovered in the epileptic phase. Per1 and Per2 rhythmic expression were disrupted in post-SE groups while Per3 presented an arrhythmic profile in the epileptic phase, only. The expression of Clock did not display rhythmic pattern in any condition. These oscillating patterns of core clock genes may contribute to hippocampal 24 h cycling and, consequently to seizure periodicity. Furthermore, by using a pool of samples collected at 6 different Zeitgeber Times (ZT), we found that all clock transcripts were significantly dysregulated after SE induction, except Per3 and Per2. Collectively, altered SLA rhythm in early post-SE and epileptic phases implies a possible role for seizure as a nonphotic cue, which is likely linked to activation of hippocampal–accumbens pathway. On the other hand, altered temporal expression of the clock genes after SE suggests their involvement in the MTLE.