Garcia-Cairasco, N. | bioRxiv Science

bioRxiv2021

Maternal behavior and the neonatal HPA axis in the Wistar Audiogenic Rat (WAR) strain: early life implications for a genetic animal model in epilepsy

Epileptogenesis is a multistage process and seizure susceptibility can be influenced by stress early in life. Wistar Audiogenic Rat (WAR) strain is an interesting model to study the association between stress and epilepsy, since it is naturally susceptible to seizures and present changes in the hypothalamus-pituitary-adrenal (HPA) axis activity. All these features are related to the pathogenic mechanisms usually associated to psychiatric comorbidities present in epilepsy. Therefore, the current study aimed to evaluate the neonate HPA axis function and maternal care under control and stress conditions in the WAR strain. Maternal behavior and neonate HPA axis were evaluated in Wistar and WAR strains under rest and after the presence of stressors. We observed that WAR pups present higher plasmatic corticosterone concentration as compared to Wistar pups. Although WAR dams do not show significant altered maternal behavior at rest, there is a higher latency to recover the litter in the pup retrieval test, while some did not recover all the litter. WAR dams presented similar behaviors to Wistar dams to a female intruder and maternal care with the pups in the maternal defense test. Taken together, these findings indicate that the WAR strain could show HPA axis disruption early in life and dams present altered maternal behavior under stressful events. Those alterations make the WAR strain an interesting model to evaluate vulnerability to epilepsy and its associated neuropsychiatric comorbidities.

neuroscience↗

bioRxiv2019

Oxytocin, Compulsion and Epilepsy: Insights from a Complex Behavioral and Neuronal Networks Association

Previously we have demonstrated that microinjection of oxytocin (OT) into the central nucleus of amygdala (CeA) induces hypergrooming in Wistar rats, a model of compulsion. The Wistar Audiogenic Rat (WAR) strain is a genetic model of generalized tonic-clonic seizures. Here we quantified grooming behavior in WAR, with grooming scores, flowcharts and directed graphs of syntactic and non-syntactic grooming chains, after bilateral administration of OT or saline (SAL) into the CeA and investigated the association between hypergrooming and imunohistochemistry of Fos activated compulsion networks and proposing a computational model of grooming behavior. The activated networks, driven from a CeA OT-dependent grooming pattern, in both Wistar and WAR were detected as Fos+ regions: orbitofrontal cortex, striatum, paraventricular nucleus of the hypothalamus, dentate gyrus, substantia nigra compacta and reticulata. In conclusion we can drive hypergrooming in WARs, defined previously as a model of ritualistic motor behavior in Wistar rats, with OT from CeA, a limbic structure and one of the principal amygdala complex outputs. Furthermore, our current pioneer behavioral and cellular description considers that hypergrooming (compulsion) in WARs is a comorbidity because: (1) WARs have the highest grooming scores, when exposed only to novelty (2) WARs have better grooming scores than Wistars after CeA-SAL, (3) WARs perform much better than Wistars in OT-CeA-dependent highly stereotyped behavioral sequences, detected by flowcharts as a combination of syntactic/non-syntactic grooming chains, (4) the behavioral sequences here demonstrated for grooming and hypergrooming can be modeled as quite reliable Markov chains and (5) with the exception of CeA-SAL injected animals, an exquisite map of brain Fos expression was detected in typical cortico-striatal-thalamic-basal ganglia-cortical circuit, among new areas, driven by OT-CeA.\n\nAuthor SummaryGrooming is a complex set of regular behavioral sequences in rodents that can be mimicked with several pharmacological or molecular biology interventions. We have demonstrated previously that microinjection of the brain peptide oxytocin into the amygdala, a limbic region, induces hypergrooming in Wistar rats, a model of compulsion. The Wistar Audiogenic Rat strain is a genetic model of generalized seizures, in fact a model of epilepsy. Here we quantified grooming behavior in Wistar Audiogenic Rats, using several behavioral tools such as grooming scores, behavioral sequences and graphs of grooming chains, after bilateral administration of the oxytocin or its control (saline) into the amygdala. We also investigated the association between hypergrooming and activation of compulsion networks, proposing a computational (virtual) model of grooming behavior. Basically we were able to detect activated networks, driven from amygdala and the consequent oxytocin-dependent grooming pattern in epileptic and control animals. Those circuits are composed of cortical and subcortical areas, usually associated to the expression of motor rituals or compulsions. In conclusion, we can drive hypergrooming in epileptic animals, as compared to the previously defined model of ritualistic/compulsive motor behavior in control, rats. We concluded that hypergrooming (compulsion) is endogenously present in epileptic animals as a co-existent event (comorbidity), because when they were exposed to novelty, they express better grooming scores than control animals. The behavioral sequences here demonstrated for grooming and hypergrooming can be simulated as chains, where associations can be predicted from probabilities. Finally, an exquisite map of brain-activated cells was detected in both epileptic animal and their controls, in typical cortico-subcortical structures associated with rituals, but driven from a region which control emotions.

neuroscience↗

bioRxiv2018

A freeze-and-thaw induced-fragment of the microtubule-associated protein Tau in rat brain extracts: implications for the study of neurodegenerative diseases

Tau is a microtubule-associated protein responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In this study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots were stored for at least 2 weeks at either -20{degrees}C or -80{degrees}C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ~25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry analysis in excised bands revealed this ~25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80{degrees}C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.

biochemistry↗