Different types of Status Epilepticus may lead to similar hippocampal epileptogenesis processes.

About 1-2% of people worldwide suffer from epilepsy, which is characterized by unpredictable and intermittent seizure occurrence. Despite the fact that the exact origin of temporal lobe epilepsy is frequently unknown, it is frequently linked to an early triggering insult like brain damage, tumors, or Status Epilepticus (SE). We used an experimental approach consisting of electrical stimulation of the amygdaloid complex to induce two behaviorally and structurally distinct SE states: Type I (fully convulsive), with more severe seizure behaviors and more extensive brain damage, and Type II (partial convulsive), with less severe seizure behaviors and brain damage. Our goal was to better understand how the various types of SE impact the hippocampus leading to the development of epilepsy. Despite clear variations between the two behaviors in terms of neurodegeneration, study of neurogenesis revealed a comparable rise in the number of Ki-67 + cells and an increase in Doublecortin (DCX) in both kinds of SE.

Audiogenic Seizures in the Streptozotocin-Induced Rat Alzheimer's Disease Model.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative and progressive disorder with no cure and constant failures in clinical trials. The main AD hallmarks are amyloid-ß (Aß) plaques, neurofibrillary tangles, and neurodegeneration. However, many other events have been implicated in AD pathogenesis. Epilepsy is a common comorbidity of AD and there is important evidence indicating a bidirectional link between these two disorders. Some studies suggest that disturbed insulin signaling might play an important role in this connection. OBJECTIVE: To understand the effects of neuronal insulin resistance in the AD-epilepsy link. METHODS: We submitted the streptozotocin (STZ) induced rat AD Model (icv-STZ AD) to an acute acoustic stimulus (AS), a known trigger of seizures. We also assessed animals' performance in the memory test, the Morris water maze and the neuronal activity (c-Fos protein) induced by a single audiogenic seizure in regions that express high levels of insulin receptors. RESULTS: We identified significant memory impairment and seizures in 71.43% of all icv-STZ/AS rats, in contrast to 22.22% of the vehicle group. After seizures, icv-STZ/AS rats presented higher number of c-Fos immunopositive cells in hippocampal, cortical, and hypothalamic regions. CONCLUSION: STZ may facilitate seizure generation and propagation by impairment of neuronal function, especially in regions that express high levels of insulin receptors. The data presented here indicate that the icv-STZ AD model might have implications not only for AD, but also for epilepsy. Finally, impaired insulin signaling might be one of the mechanisms by which AD presents a bidirectional connection to epilepsy.

Cannabidiol modulates chronic neuropathic pain aversion behavior by attenuation of neuroinflammation markers and neuronal activity in the corticolimbic circuit in male Wistar rats.

Chronic neuropathic pain (CNP) is a vast world health problem often associated with the somatosensory domain. This conceptualization is problematic because, unlike most other sensations that are usually affectively neutral and may present emotional, affective, and cognitive impairments. Neuronal circuits that modulate pain can increase or decrease painful sensitivity based on several factors, including context and expectation. The objective of this study was to evaluate whether subchronic treatment with Cannabidiol (CBD; 0.3, 3, and 10 mg/kg intraperitoneal route - i.p., once a day for 3 days) could promote pain-conditioned reversal, in the conditioned place preference (CPP) test, in male Wistar rats submitted to chronic constriction injury (CCI) of the sciatic nerve. Then, we evaluated the expression of astrocytes and microglia in animals treated with CBD through the immunofluorescence technique. Our results demonstrated that CBD promoted the reversal of CPP at 3 and 10 mg/kg. In CCI animals, CBD was able to attenuate the increase in neuronal hyperactivity, measured by FosB protein expression, in the regions of the corticolimbic circuit: anterior cingulate cortex (ACC), complex basolateral amygdala (BLA), granular layer of the dentate gyrus (GrDG), and dorsal hippocampus (DH) - adjacent to subiculum (CA1). CBD also prevented the increased expression of GFAP and IBA-1 in CCI animals. We concluded that CBD effects on CNP are linked to the modulation of the aversive component of pain. These effects decrease chronic neuronal activation and inflammatory markers in regions of the corticolimbic circuit.

Evidence of disturbed insulin signaling in animal models of Alzheimer's disease.

Since glucose reuptake by neurons is mostly independent of insulin, it has been an intriguing question whether insulin has or not any roles in the brain. Consequently, the identification of insulin receptors in the central nervous system has fueled investigations of insulin functions in the brain. It is also already known that insulin can influence glucose reuptake by neurons, mostly during activities that have the highest energy demand. The identification of high density of insulin receptors in the hippocampus also suggests that insulin may present important roles related to memory. In this context, studies have reported worse performance in cognitive tests among diabetic patients. In addition, alterations in the regulation of central insulin pathways have been observed in the brains of Alzheimer's disease (AD) patients. In fact, some authors have proposed AD as a third type of diabetes and recently, our group proposed insulin resistance as a common link between different AD hypotheses. Therefore, in the present narrative review, we intend to revise and gather the evidence of disturbed insulin signaling in experimental animal models of AD.

The resilience of adolescent male rats to acute stress-induced delayed anxiety is age-related and glucocorticoid release-dependent.

Studies investigated how stressful experiences modulate physiological and behavioral responses and the consequences of stress-induced corticosterone release in anxiety-like behavior. Adolescence is crucial to brain maturation, and several neurobiological changes in this period lead individuals to increased susceptibility or resilience to aversive situations. Despite the effects of stress in adults, information about adolescents' responses to acute stress is lacking. We aimed to understand how adolescence affects acute stress responses. Male adolescent rats (30 days old) were 2 h restrained, and anxiety-like behaviors were measured immediately or 10 days after stress in the elevated plus-maze (EPM) and the light-dark box (LDB) tests. To verify the importance of CORT modulation in stress-induced anxiety, another group of rats was treated, 30 min before restraint, with metyrapone to blunt the stress-induced CORT peak and tested immediately after stress. To show that stress effects on behavior were age-dependent, another set of rats was tested in two different periods - early adolescence (30 days old) and mid-adolescence (40 days old) and were treated or not with metyrapone before the stress session and tested immediately or ten days later in the LDB test. Only early adolescent male rats were resilient to delayed anxiety-like behavior in EPM and LDB tests. Metyrapone treatment increased the rats' exploration immediately and ten days after stress. These data suggest a specific age at which adolescent rats are resilient to the delayed effects of acute restraint stress and that the metyrapone treatment has long-term behavioral consequences.

Behavior and electrophysiological effects on striatum-nigra circuit after high frequency stimulation. Relevance to Parkinson and epilepsy.

The phenomenon of plasticity in the striatum, and its relation with the striatum-nigra neuronal circuit has clinical and neurophysiological relevance to Parkinson and epilepsy. High frequency stimulation (HFS) can induce neural plasticity. Furthermore, it is possible to induce plasticity in the dorsal striatum and this can be modulated by substantia nigra activity. But it has not been shown yet what would be the effects in the striatum-nigra circuit after plasticity induction in striatum with HSF. Literature also misses a detailed description of the way back loop of the circuit: the striatal firing rate after substantia nigrás inhibition. We here conducted: First Experiment, application of HFS in dorsomedial striatum and measure of spontaneous and longlasting behavior expression in the open field three days later; Second, application of single pulses on dorsomedial striatum and measure of the evoked potentials in substantia nigra before and after HFS; Third Experiment: inhibition of substantia nigra and recording of the firing rate of dorsomedial striatum. HFS in dorsomedial striatum caused increased locomotion behaviors, but not classical stereotypy. However, rats had either an increase or decrease in substantia nigrás evoked potentials. Also, substantia nigrás inhibition caused an increase in dorsomedial striatum firing rate. Present data are suggestive of a potential application of HFS in striatum, as an attempt to modulate behavior rigidity and hypokinesia of diseases involving the basal ganglia, especially Parkinson´s Disease.

Neuroprotective Effect of Exogenous Galectin-1 in Status Epilepticus.

Intrahippocampal pilocarpine microinjection (H-PILO) induces status epilepticus (SE) that can lead to spontaneous recurrent seizures (SRS) and neurodegeneration in rodents. Studies using animal models have indicated that lectins mediate a variety of biological activities with neuronal benefits, especially galectin-1 (GAL-1), which has been identified as an effective neuroprotective compound. GAL-1 is associated with the regulation of cell adhesion, proliferation, programmed cell death, and immune responses, as well as attenuating neuroinflammation. Here, we administrated GAL-1 to Wistar rats and evaluated the severity of the SE, neurodegenerative and inflammatory patterns in the hippocampal formation. Administration of GAL-1 caused a reduction in the number of class 2 and 4 seizures, indicating a decrease in seizure severity. Furthermore, we observed a reduction in inflammation and neurodegeneration 24 h and 15 days after SE. Overall, these results suggest that GAL-1 has a neuroprotective effect in the early stage of epileptogenesis and provides new insights into the roles of exogenous lectins in temporal lobe epilepsy (TLE).

Challenges in the use of animal models and perspectives for a translational view of stress and psychopathologies.

The neurobiology and development of treatments for stress-related neuropsychiatric disorders rely heavily on animal models. However, the complexity of these disorders makes it difficult to model them entirely, so only specific features of human psychopathology are emulated and these models should be used with great caution. Importantly, the effects of stress depend on multiple factors, like duration, context of exposure, and individual variability. Here we present a review on pre-clinical studies of stress-related disorders, especially those developed to model posttraumatic stress disorder, major depression, and anxiety. Animal models provide relevant evidence of the underpinnings of these disorders, as long as face, construct, and predictive validities are fulfilled. The translational challenges faced by scholars include reductionism and anthropomorphic/anthropocentric interpretation of the results instead of a more naturalistic and evolutionary understanding of animal behavior that must be overcome to offer a meaningful model. Other limitations are low statistical power of analysis, poor evaluation of individual variability, sex differences, and possible conflicting effects of stressors depending on specific windows in the lifespan.

A Genetic Model of Epilepsy with a Partial Alzheimer's Disease-Like Phenotype and Central Insulin Resistance.

Studies have suggested an important connection between epilepsy and Alzheimer's disease (AD), mostly due to the high number of patients diagnosed with AD who develop epileptic seizures later on. However, this link is not well understood. Previous studies from our group have identified memory impairment and metabolic abnormalities in the Wistar audiogenic rat (WAR) strain, a genetic model of epilepsy. Our goal was to investigate AD behavioral and molecular alterations, including brain insulin resistance, in naïve (seizure-free) animals of the WAR strain. We used the Morris water maze (MWM) test to evaluate spatial learning and memory performance and hippocampal tissue to verify possible molecular and immunohistochemical alterations. WARs presented worse performance in the MWM test (p < 0.0001), higher levels of hyperphosphorylated tau (S396) (p < 0.0001) and phosphorylated glycogen synthase kinase 3 (S21/9) (p < 0.05), and lower insulin receptor levels (p < 0.05). Conversely, WARs and Wistar controls present progressive increase in amyloid fibrils (p < 0.0001) and low levels of soluble amyloid-ß. Interestingly, the detected alterations were age-dependent, reaching larger differences in aged than in young adult animals. In summary, the present study provides evidence of a partial AD-like phenotype, including altered regulation of insulin signaling, in a genetic model of epilepsy. Together, these data contribute to the understanding of the connection between epilepsy and AD as comorbidities. Moreover, since both tau hyperphosphorylation and altered insulin signaling have already been reported in epilepsy and AD, these two events should be considered as important components in the interconnection between epilepsy and AD pathogenesis and, therefore, potential therapeutic targets in this field.

A complex systems view on the current hypotheses of epilepsy pharmacoresistance.

Drug-resistant epilepsy remains to this day as a highly prevalent condition affecting around one-third of patients with epilepsy, despite all the research and the development of several new antiseizure medications (ASMs) over the last decades. Epilepsies are multifactorial complex diseases, commonly associated with psychiatric, neurological, and somatic comorbidities. Thus, to solve the puzzling problem of pharmacoresistance, the diagnosis and modeling of epilepsy and comorbidities need to change toward a complex system approach. In this review, we have summarized the sequence of events for the definition of epilepsies and comorbidities, the search for mechanisms, and the major hypotheses of pharmacoresistance, drawing attention to some of the many converging aspects between the proposed mechanisms, their supporting evidence, and comorbidities-related alterations. The use of systems biology applied to epileptology may lead to the discovery of new targets and the development of new ASMs, as may advance our understanding of the epilepsies and their comorbidities, providing much deeper insight on multidrug pharmacoresistance.

Increased TRPV1 Channels and FosB Protein Expression Are Associated with Chronic Epileptic Seizures and Anxiogenic-like Behaviors in a Preclinical Model of Temporal Lobe Epilepsy.

Epilepsies are neurological disorders characterized by chronic seizures and their related neuropsychiatric comorbidities, such as anxiety. The Transient Receptor Potential Vanilloid type-1 (TRPV1) channel has been implicated in the modulation of seizures and anxiety-like behaviors in preclinical models. Here, we investigated the impact of chronic epileptic seizures in anxiety-like behavior and TRPV1 channels expression in a genetic model of epilepsy, the Wistar Audiogenic Rat (WAR) strain. WARs were submitted to audiogenic kindling (AK), a preclinical model of temporal lobe epilepsy (TLE) and behavioral tests were performed in the open-field (OF), and light-dark box (LDB) tests 24 h after AK. WARs displayed increased anxiety-like behavior and TRPV1R expression in the hippocampal CA1 area and basolateral amygdala nucleus (BLA) when compared to control Wistar rats. Chronic seizures increased anxiety-like behaviors and TRPV1 and FosB expression in limbic and brainstem structures involved with epilepsy and anxiety comorbidity, such as the hippocampus, superior colliculus, and periaqueductal gray matter. Therefore, these results highlight previously unrecognized alterations in TRPV1 expression in brain structures involved with TLE and anxiogenic-like behaviors in a genetic model of epilepsy, the WAR strain, supporting an important role of TRPV1 in the modulation of neurological disorders and associated neuropsychiatric comorbidities.

Absence epilepsy in male and female WAG/Rij rats: A longitudinal EEG analysis of seizure expression.

The WAG/Rij strain of rats is commonly used as a preclinical model of genetic absence epilepsy. While widely utilized, the developmental trajectory of absence seizure expression has been only partially described. Moreover, sex differences in this strain have been under-explored. Here, we longitudinally monitored male and female WAG/Rij rats to quantify cortical spike-and-wave discharges (SWDs) monthly, from 4 to 10 months of age. In both male and female WAG/Rij rats, absence seizure susceptibility increased with age. In contrast to previous reports, we found a robust and consistent increase in absence epilepsy susceptibility in male WAG/Rij rats in comparison to females across months. The increased absence seizure susceptibility was characterized by increased number and duration of SWDs, and consequently increased total SWDs duration. These findings highlight a previously un-recognized sex difference in a model of absence epilepsy and narrow the knowledge gap of age-dependent expression of SWDs in the WAG/Rij strain.

Cannabidiol effectively reverses mechanical and thermal allodynia, hyperalgesia, and anxious behaviors in a neuropathic pain model: Possible role of CB1 and TRPV1 receptors.

The incidence of chronic pain is high in the general population and it is closely related to anxiety disorders, which promote negative effects on the quality of life. The cannabinoid system has essential participation in the pain sensitivity circuit. In this perspective, cannabidiol (CBD) is considered a promising strategy for treating neuropathic pain. Our study aimed to evaluate the effects of sub-chronic systemic treatment with CBD (0.3, 3, 10, or 30 mg/kg, i.p.) in male in rats submitted to chronic constriction injury of the sciatic nerve (CCI) or not (SHAM) and assessed in nociceptive tests (von Frey, acetone, and hot plate, three days CBD's treatment) and in the open field test (OFT, two days CBD's treatment). We performed a screening immunoreactivity of CB1 and TRPV1 receptors in cortical and limbic regions tissues, which were collected after 1.5 h of behavioral tests on the 24th experimental day. This study presents a dose-response curve to understand better the effects of low doses (3 mg/kg) on CBD's antiallodynic and anxiolytic effects. Also, low doses of CBD were able to (1) reverse mechanical and thermal allodynia (cold) and hyperalgesia, (2) reverse anxious behaviors (reduction of the % of grooming and freezing time, and increase of the % of center time in the OFT) induced by chronic pain. The peripheral neuropathy promoted the increase in the expression of CB1 and TRPV1 receptors in the anterior cingulate cortex (ACC), anterior insular cortex (AIC), basolateral amygdala (BLA), dorsal hippocampus (DH), and ventral hippocampus (VH). CBD potentiated this effect in the ACC, AIC, BLA, DH, and VH regions. These results provide substantial evidence of the role of the ACC-AIC-BLA corticolimbic circuit, and BLA-VH for pain regulation. These results can be clinically relevant since they contribute to the evidence of CBD's beneficial effects on treating chronic pain and associated comorbidities such as anxiety.

Putative Causal Variant on Vlgr1 for the Epileptic Phenotype in the Model Wistar Audiogenic Rat.

Wistar Audiogenic Rat is an epilepsy model whose animals are predisposed to develop seizures induced by acoustic stimulation. This model was developed by selective reproduction and presents a consistent genetic profile due to the several generations of inbreeding. In this study, we performed an analysis of WAR RNA-Seq data, aiming identified at genetic variants that may be involved in the epileptic phenotype. Seventeen thousand eighty-five predicted variants were identified as unique to the WAR model, of which 15,915 variants are SNPs and 1,170 INDELs. We filter the predicted variants by pre-established criteria and selected five for validation by Sanger sequencing. The genetic variant c.14198T>C in the Vlgr1 gene was confirmed in the WAR model. Vlgr1 encodes an adhesion receptor that is involved in the myelination process, in the development of stereocilia of the inner ear, and was already associated with the audiogenic seizures presented by the mice Frings. The transcriptional quantification of Vlgr1 revealed the downregulation this gene in the corpus quadrigeminum of WAR, and the protein modeling predicted that the mutated residue alters the structure of a domain of the VLGR1 receptor. We believe that Vlgr1 gene may be related to the predisposition of WAR to seizures and suggest the mutation Vlgr1/Q4695R as putative causal variant, and the first molecular marker of the WAR strain.

Insulin Resistance as a Common Link Between Current Alzheimer's Disease Hypotheses.

Almost 115 years ago, Alois Alzheimer described Alzheimer's disease (AD) for the first time. Since then, many hypotheses have been proposed. However, AD remains a severe health public problem. The current medical approaches for AD are limited to symptomatic interventions and the complexity of this disease has led to a failure rate of approximately 99.6%in AD clinical trials. In fact, no new drug has been approved for AD treatment since 2003. These failures indicate that we are failing in mimicking this disease in experimental models. Although most studies have focused on the amyloid cascade hypothesis of AD, the literature has made clear that AD is rather a multifactorial disorder. Therefore, the persistence in a single theory has resulted in lost opportunities. In this review, we aim to present the striking points of the long scientific path followed since the description of the first AD case and the main AD hypotheses discussed over the last decades. We also propose insulin resistance as a common link between many other hypotheses.

Neuroendocrine changes in the hypothalamic-neurohypophysial system in the Wistar audiogenic rat (WAR) strain submitted to audiogenic kindling.

The Wistar audiogenic rat (WAR) strain is used as an animal model of epilepsy, which when submitted to acute acoustic stimulus presents tonic-clonic seizures, mainly dependent on brainstem (mesencephalic) structures. However, when WARs are exposed to chronic acoustic stimuli (audiogenic kindling-AK), they usually present tonic-clonic seizures, followed by limbic seizures, after recruitment of forebrain structures such as the cortex, hippocampus and amygdala. Although some studies have reported that hypothalamic-hypophysis function is also altered in WAR through modulating vasopressin (AVP) and oxytocin (OXT) secretion, the role of these neuropeptides in epilepsy still is controversial. We analyzed the impact of AK and consequent activation of mesencephalic neurocircuits and the recruitment of forebrain limbic (LiR) sites on the hypothalamic-neurohypophysial system and expression of Avpr1a and Oxtr in these structures. At the end of the AK protocol, nine out of 18 WARs presented LiR. Increases in both plasma vasopressin and oxytocin levels were observed in WAR when compared to Wistar rats. These results were correlated with an increase in the expressions of heteronuclear (hn) and messenger (m) RNA for Oxt in the paraventricular nucleus (PVN) in WARs submitted to AK that presented LiR. In the paraventricular nucleus, the hnAvp and mAvp expressions increased in WARs with and without LiR, respectively. There were no significant differences in Avp and Oxt expression in supraoptic nuclei (SON). Also, there was a reduction in the Avpr1a expression in the central nucleus of the amygdala and frontal lobe in the WAR strain. In the inferior colliculus, Avpr1a expression was lower in WARs after AK, especially those without LiR. Our results indicate that both AK and LiR in WARs lead to changes in the hypothalamic-neurohypophysial system and its receptors, providing a new molecular basis to better understaind epilepsy.

Cannabinoids in Audiogenic Seizures: From Neuronal Networks to Future Perspectives for Epilepsy Treatment.

Cannabinoids and Cannabis-derived compounds have been receiving especial attention in the epilepsy research scenario. Pharmacological modulation of endocannabinoid system's components, like cannabinoid type 1 receptors (CB1R) and their bindings, are associated with seizures in preclinical models. CB1R expression and functionality were altered in humans and preclinical models of seizures. Additionally, Cannabis-derived compounds, like cannabidiol (CBD), present anticonvulsant activity in humans and in a great variety of animal models. Audiogenic seizures (AS) are induced in genetically susceptible animals by high-intensity sound stimulation. Audiogenic strains, like the Genetically Epilepsy Prone Rats, Wistar Audiogenic Rats, and Krushinsky-Molodkina, are useful tools to study epilepsy. In audiogenic susceptible animals, acute acoustic stimulation induces brainstem-dependent wild running and tonic-clonic seizures. However, during the chronic protocol of AS, the audiogenic kindling (AuK), limbic and cortical structures are recruited, and the initially brainstem-dependent seizures give rise to limbic seizures. The present study reviewed the effects of pharmacological modulation of the endocannabinoid system in audiogenic seizure susceptibility and expression. The effects of Cannabis-derived compounds in audiogenic seizures were also reviewed, with especial attention to CBD. CB1R activation, as well Cannabis-derived compounds, induced anticonvulsant effects against audiogenic seizures, but the effects of cannabinoids modulation and Cannabis-derived compounds still need to be verified in chronic audiogenic seizures. The effects of cannabinoids and Cannabis-derived compounds should be further investigated not only in audiogenic seizures, but also in epilepsy related comorbidities present in audiogenic strains, like anxiety, and depression.

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 with 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. Wistar Audiogenic Rat 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.

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity.

Tau is a microtubule-associated protein (MAP) 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 the present 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 of the same extracts were stored for at least 2 weeks at either -20 or -80°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 (MS) 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°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.

Evaluation of the HPA Axis' Response to Pharmacological Challenges in Experimental and Clinical Early-Life Stress-Associated Depression.

Early-life stress (ELS) is associated with a higher risk of psychopathologies in adulthood, such as depression, which may be related to persistent changes in the hypothalamic-pituitary-adrenal (HPA) axis. This study aimed to evaluate the effects of ELS on the functioning of the HPA axis in clinical and experimental situations. Clinically, patients with current depressive episodes, with and without ELS, and healthy controls, composed the sample. Subjects took a capsule containing placebo, fludrocortisone, prednisolone, dexamethasone or spironolactone followed by an assessment of plasma cortisol the morning after. Experimentally, male Wistar rats were submitted to ELS protocol based on variable, unpredictable stressors from postnatal day (PND)1 to PND21. On PND65 animals were behaviorally evaluated through the forced-swimming test (FST). At PND68, pharmacological challenges started, using mifepristone, dexamethasone, spironolactone, or fludrocortisone, and corticosterone levels were determined 3 h after injections. Cortisol response of the patients did not differ significantly from healthy subjects, regardless of their ELS history, and it was lower after fludrocortisone, prednisolone, and dexamethasone compared with placebo, indicating the suppression of plasma cortisol by all these treatments. Animals exposed to ELS presented altered phenotype as indicated by an increased immobility time in the FST when compared with control, but no significant long-lasting effects of ELS were observed on the HPA axis response. Limitations on the way the volunteers were sampled may have contributed to the lack of ELS effects on the HPA axis, pointing out the need for further research to understand these complex phenomena.