Wistar Audiogenic Rat Research Articles

Changes in autonomic control of the cardiovascular system in the Wistar audiogenic rat (WAR) strain

We evaluated autonomic cardiovascular modulation and baroreflex control of heart rate (HR) in a particular epileptic rat strain, Wistar audiogenic rats (WARs). We studied spontaneous baroreflex sensitivity as well as reflex changes in HR evoked by phenylephrine/nitroprusside-induced changes in arterial pressure (AP). Atropine and propranolol were used to measure cardiac autonomic tone. AP and pulse interval (PI) variability analysis were performed in the time and frequency domains (FFT spectral analysis) to evaluate cardiovascular sympatovagal modulation in WARs. AP and HR were higher in WARs (109±2mm Hg and 366±9bpm) than in Wistar control rats (101±2mm Hg and 326±10bpm). The power of the low-frequency band of both AP and PI spectra, a marker of sympathetic modulation, was higher in WARs than in Wistar control rats. The high-frequency power of the PI spectra in normalized units, which is linked to cardiac vagal modulation, was lower in WARs. Both WARs and Wistar control rats had similar vagal tone (91±13bpm vs 94±11bpm, respectively), but sympathetic tone was higher in WARs (30±4bpm vs 14±4bpm). No differences were detected in the gain of evoked (1.32±0.1ms/mm Hg vs 1.35±0.2ms/mm Hg) or spontaneous (1.34±0.2ms/mm Hg vs 2.04±0.2ms/mm Hg) baroreflex sensitivity. The higher AP and HR and the autonomic imbalance (sympathetic predominance) in WARs might be associated with an increased risk of life-threatening cardiovascular events in this strain.

Reduced hippocampal GABAergic function in Wistar audiogenic rats

Epilepsy is a neurological disorder associated with excitatory and inhibitory imbalance within the underlying neural network. This study evaluated inhibitory γ-amino-butyric acid (GABA)ergic modulation in the CA1 region of the hippocampus of male Wistar rats and Wistar audiogenic rats (aged 90 ± 3 days), a strain of inbred animals susceptible to audiogenic seizures. Field excitatory postsynaptic potentials and population spike complexes in response to Schaffer collateral fiber stimulation were recorded in hippocampal slices before and during application of picrotoxin (50 µM, 60 min), a GABA A antagonist, and the size of the population spike was quantified by measuring its amplitude and slope. In control audiogenic-resistant Wistar rats (N = 9), picrotoxin significantly increased both the amplitude of the population spike by 51 ± 19% and its maximum slope by 73 ± 21%. In contrast, in slices from Wistar audiogenic rats (N = 6), picrotoxin caused no statistically significant change in population spike amplitude (33 ± 46%) or slope (11 ± 29%). Data are reported as means ± SEM. This result indicates a functional reduction of GABAergic neurotransmission in hippocampal slices from Wistar audiogenic rats.

Behavioral and EEG effects of GABAergic manipulation of the nigrotectal pathway in the Wistar audiogenic rat strain

The superior colliculus (SC), substantia nigra pars reticulata (SNPr), and striatum have been characterized as important structures involved in the modulation of seizure activity. In the current study, bicuculline (GABAA antagonist) and muscimol (GABAA agonist) were microinjected into the deep layers of either the anterior SC (aSC) or posterior SC (pSC) of genetically developed Wistar audiogenic rats. Behavior and EEG activity were studied simultaneously. Only muscimol microinjected into the pSC had behavioral and EEG anticonvulsant effects in Wistar audiogenic rats, eliciting EEG oscillation changes in both SNPr and pSC, primarily during tonic seizures. The SC of Wistar audiogenic rats thus comprises two functionally different subregions, pSC and aSC, defined by distinct behavioral and EEG features. The pSC has proconvulsant audiogenic seizure activity in Wistar audiogenic rats. Our data suggest that this phenomenon may be a consequence of the genetic selection of the Wistar audiogenic rat strain.

Functional characterization of the hypothalamic–pituitary–adrenal axis of the Wistar Audiogenic Rat (WAR) strain

The Wistar Audiogenic Rat (WAR) strain is a genetic model of sound-induced reflex epilepsy which was selected starting from audiogenic seizures susceptible Wistar rats. Wistar resistant rats were used as WAR's control in this study. In the acute situation, audiogenic seizures (AS) in WARs mimic tonic–clonic seizures and, in the chronic protocol, mimic temporal lobe epilepsy. AS have been shown to evoke neuroendocrine responses; however, the hypothalamic–pituitary–adrenal activity in the WAR has not been established. The aim of this study was to evaluate the hypothalamic–pituitary–adrenal axis (HPA) responses to exogenous ACTH stimulation (8ng/rat), fifteen minute restraint stress and circadian variation (8am and 8pm) under rest conditions in these animals through plasma measurements of ACTH and corticosterone concentrations. We also measured the body weight from birth to the 9th week of life and determined adrenal gland weight. We found that WARs are smaller than Wistar and presented a higher adrenal gland weight with a higher level of corticosterone release after intravenous ACTH injection. They also showed altered HPA axis circadian rhythms and responses to restraint stress. Our data indicate that, despite the lower body weight, WARs have increased adrenal gland weight associated with enhanced pituitary and adrenal responsiveness after HPA axis stimulation. Thus, we propose WARs as a model to study stress–epilepsy interactions and epilepsy–neuropsychiatry comorbidities.

The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling

The aim of this study was to identify molecular pathways involved in audiogenic seizures in the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling) when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1−/− mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs.

Changes in autonomic control of the cardiovascular system in the Wistar audiogenic rat (WAR) strain, an experimental model of epilepsy

This study aimed to evaluate autonomic cardiovascular modulation and baroreflex control of heart rate (HR) in Wistar audiogenic rats (WAR), an epileptic rat strain. We studied spontaneous baroreflex (sequence method) as well as reflex changes in HR evoked by phenylephrine or nitroprusside‐induced changes in arterial pressure (AP). Blockers of autonomic receptors (atropine and propranolol) were used to measure cardiac autonomic tone. Pulse interval (PI) and AP variability analyses were performed by FFT spectral analysis to evaluate cardiovascular sympathovagal modulation. In WAR, AP and HR were higher (109±2 mmHg and 366±9 bpm) as compared with Wistar rats (101±2 mmHg and 326±10 bpm). Power in the low frequency (0.2–0.8 Hz) band, a marker of sympathetic activity, was higher in the AP and PI spectra of WAR than in those of Wistar rats. High frequency (0.8–3 Hz) power of PI spectra, which is linked to cardiac vagal modulation, was lower in WAR. Both strains showed similar vagal tone (91±13 vs. 94±11 bpm) but sympathetic tone was higher in WAR (30±4 vs. 14±4 bpm). No differences were found in the gains of evoked (1.32±0.1 vs. 1.35±0.2 ms/mmHg) or spontaneous (1.34±0.2 vs. 2.04±0.2 ms/mmHg) baroreflex sensitivity. Higher AP and HR are well known to be associated with sympathetic predominance and with threatening cardiovascular events, which are typical causes of sudden unexpected death in epilepsy. Support: FAPESP, CNPq.

Role of the superior colliculus in the expression of acute and kindled audiogenic seizures in Wistar audiogenic rats

The role of the superior colliculus (SC) in seizure expression is controversial and appears to be dependent upon the epilepsy model. This study shows the effect of disconnection between SC deep layers and adjacent tissues in the expression of acute and kindling seizures. Subcollicular transections, ablation of SC superficial and deep layers, and ablation of only the cerebral cortex were evaluated in the Wistar audiogenic rat (WAR) strain during acute and kindled audiogenic seizures. The audiogenic seizure kindling protocol started 4 days after surgeries, with two acoustic stimuli per day for 10 days. Acute audiogenic seizures were evaluated by a categorized seizure severity midbrain index (cSI) and kindled seizures by a severity limbic index (LI). All subcollicular transections reaching the deep layers of the SC abolished audiogenic seizures or significantly decreased cSI. In the unlesioned kindled group, a reciprocal relationship between limbic and brainstem pattern of seizures was seen. The increased number of stimuli provoked an audiogenic kindling phenomenon. Ablation of the entire SC (ablation group) or of the cerebral cortex only (ctx-operated group) hampered the acquisition of limbic behaviors. There was no difference in cSI and LI between the ctx-operated and ablation groups, but there was a difference between ctx-operated and the unlesioned kindled group. There was also no difference in cSI between SC deep layer transection and ablation groups. Results of histologic analyses were similar for acute and kindled audiogenic seizure groups. SC deep layers are involved in the expression of acute and kindled audiogenic seizure, and the cerebral cortex is essential for audiogenic kindling development.

Increased expression of GluR2‐flip in the hippocampus of the Wistar audiogenic rat strain after acute and kindled seizures

The Wistar Audiogenic Rat (WAR) is an epileptic-prone strain developed by genetic selection from a Wistar progenitor based on the pattern of behavioral response to sound stimulation. Chronic acoustic stimulation protocols of WARs (audiogenic kindling) generate limbic epileptogenesis, confirmed by ictal semiology, amygdale, and hippocampal EEG, accompanied by hippocampal and amygdala cell loss, as well as neurogenesis in the dentate gyrus (DG). In an effort to identify genes involved in molecular mechanisms underlying epileptic process, we used suppression-subtractive hybridization to construct normalized cDNA library enriched for transcripts expressed in the hippocampus of WARs. The most represented gene among the 133 clones sequenced was the ionotropic glutamate receptor subunit II (GluR2), a member of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleopropionic acid (AMPA) receptor. Although semiquantitative RT-PCR analysis shows that the hippocampal levels of the GluR2 subunits do not differ between naïve WARs and their Wistar counterparts, we observed that the expression of the transcript encoding the splice-variant GluR2-flip is increased in the hippocampus of WARs submitted to both acute and kindled audiogenic seizures. Moreover, using in situ hybridization, we verified upregulation of GluR2-flip mainly in the CA1 region, among the hippocampal subfields of audiogenic kindled WARs. Our findings on differential upregulation of GluR2-flip isoform in the hippocampus of WARs displaying audiogenic seizures is original and agree with and extend previous immunohistochemical for GluR2 data obtained in the Chinese P77PMC audiogenic rat strain, reinforcing the association of limbic AMPA alterations with epileptic seizures.

Sexual differentiation of cortical spreading depression propagation after acute and kindled audiogenic seizures in the Wistar audiogenic rat (WAR)

Brain excitability diseases like epilepsy constitute one factor that influences brain electrophysiological features. Cortical spreading depression (CSD) is a phenomenon that can be altered by changes in brain excitability. CSD propagation was presently characterized in adult male and female rats from a normal Wistar strain and from a genetically audiogenic seizure-prone strain, the Wistar audiogenic rat (WAR), both previously submitted (RAS(+)), or not (RAS(-)), to repetitive acoustic stimulation, to provoke audiogenic kindling in the WAR-strain. A gender-specific change in CSD-propagation was found. Compared to seizure-resistant animals, in the RAS(-) condition, male and female WARs, respectively, presented CSD-propagation impairment and facilitation, characterized, respectively, by lower and higher propagation velocities (P<0.05). In contraposition, in the RAS(+) condition, male and female WARs displayed, respectively, higher and lower CSD-propagation rates, as compared to the corresponding controls. In some Wistar and WAR females, we determined estrous cycle status on the day of the CSD-recording as being either estrous or diestrous; no cycle-phase-related differences in CSD-propagation velocities were detected. In contrast to other epilepsy models, such as Status Epilepticus induced by pilocarpine, despite the CSD-velocity reduction, in no case was CSD propagation blocked in WARs. The results suggest a gender-related, estrous cycle-phase-independent modification in the CSD-susceptibility of WAR rats, both in the RAS(+) and RAS(-) situation.

The role of the inherited genetic background on the consequences of lithium-pilocarpine status epilepticus: Study in Genetic Absence Epilepsy Rats from Strasbourg and Wistar audiogenic rats

The susceptibility of rats with genetically inherited epilepsy to the genesis and consequences of secondary temporal lobe epilepsy is unknown. Here, we induced lithium-pilocarpine status epilepticus (SE) in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) or in Wistar audiogenic sensitive (AS) rats. Wistar AS needed less pilocarpine than GAERS and Non-Epileptic Rats (NERs) to develop SE. Sixty six, 40 and 5% of Wistar AS, GAERS and NERs, respectively, died within 24 h after SE. In GAERS, SE prevented the occurrence of absence seizures for 5 days. Thereafter a limited number of absence seizures with low amplitude and short duration were recorded. Wistar AS developed limbic epilepsy within 9 days after SE while GAERS and NERs needed 36–39 days to develop spontaneous motor seizures. Neuronal loss consecutive to SE was similar in the three strains and particularly marked in limbic forebrain and parahippocampal cortices. In conclusion, the development of focal limbic epilepsy in GAERS largely impairs the expression of absence seizures. The genetic background underlying the expression of audiogenic seizures sensitizes strongly the rats to a further insult and compromises their survival.

Peripheral glucose metabolism is altered by epileptic seizures

The aim of the present study was to investigate the status of jejunal absorption and peripheral metabolism of glucose in Wistar Audiogenic Rats (WAR), a genetic model of epilepsy, after seizures induced by intensive sound exposure. The jejunal loop of rats was isolated and infused (0.5 mL min(-1)) with Tyrode solution containing twice the normal concentrations of glucose, sodium, and potassium. Samples were taken at 5 or 10-min intervals over a 40-min period. At the end of the experiment, samples of liver and gastrocnemius muscle were taken to measure the levels of glycogen, glucose-6-phosphate, fructose-6-phosphate and glucose transporter-4 (GLUT4). Hepatic glucose-6-phosphate increased in WAR submitted to audiogenic seizure (21.90 +/- 3.08) as compared to non-susceptible Wistar rats (8.12 +/- 0.87) and to WAR not submitted to audiogenic stimulation (5.17 +/- 0.97). In addition, an increase in hepatic fructose-6-phosphate, an intermediate metabolite of the glycolytic pathway, was observed in WAR submitted to audiogenic seizure (5.98 +/- 0.99) compared to non-susceptible Wistar rats (2.38 +/- 0.53). According to the present results, jejunal absorption of glucose was not changed by seizures. However, generalized tonic-clonic seizures produced by sound stimulation resulted in a decrease in muscle glycogen content. In addition, our results demonstrated that the concentration of GLUT4 in the gastrocnemius muscle of WAR was 1.6-fold higher than that observed in resistant rats and that the audiogenic stimulus led to decreased concentration of this receptor in the muscle of WAR animals.

Modulation of B1 and B2 kinin receptors expression levels in the hippocampus of rats after audiogenic kindling and with limbic recruitment, a model of temporal lobe epilepsy

Epileptic seizures are hypersynchronous, paroxystic and abnormal neuronal discharges. Epilepsies are characterized by diverse mechanisms involving alteration of excitatory and inhibitory neurotransmission that result in hyperexcitability of the central nervous system (CNS). Enhanced neuronal excitability can also be achieved by inflammatory processes, including the participation of cytokines, prostaglandins or kinins, molecules known to be involved in either triggering or in the establishment of inflammation. Multiple inductions of audiogenic seizures in the Wistar audiogenic rat (WAR) strain are a model of temporal lobe epilepsy (TLE), due to the recruitment of limbic areas such as hippocampus and amygdala. In this study we investigated the modulation of the B1 and B2 kinin receptors expression levels in neonatal WARs as well as in adult WARs subjected to the TLE model. The expression levels of pro-inflammatory (IL-1β) and anti-inflammatory (IL-10) cytokines were also evaluated, as well as cyclooxygenase (COX-2). Our results showed that the B1 and B2 kinin receptors mRNAs were up-regulated about 7- and 4-fold, respectively, in the hippocampus of kindled WARs. On the other hand, the expressions of the IL-1β, IL-10 and COX-2 were not related to the observed increase of expression of kinin receptors. Based on those results we believe that the B1 and B2 kinin receptors have a pivotal role in this model of TLE, although their participation is not related to an inflammatory process. We believe that kinin receptors in the CNS may act in seizure mechanisms by participating in a specific kininergic neurochemical pathway.

Presence of endothelium prevents contraction induced by phenylephrine (PHE) in aorta from Wistar Audiogenic Rats (WARs) when the sarcoplasmic Ca2+‐ATPase (SERCA) is inhibited.

Doretto et al, (Behavior Genetics, 33:–42, 2003) developed an audiogenic seizure susceptible strain (WARs). Seizures are often associated to cardiovascular alterations. In order to understand alterations in vascular reactivity in WARs submitted to acoustic stimulation, we have studied Ca2+ pumping by SERCA and the contraction stimulated with PHE in aorta rings in the absence (E−) and in the presence of endothelium (E+). Startle induced by acoustic pulses was intercalated 24 h between acute and chronic acoustic stimuli, the latter to test audiogenic seizures. Aorta rings were isolated from acoustically stimulated male Wistar and WARs and placed into bath chambers with Krebs Salt Solution (KSS). Contraction induced by 0.3 μM PHE was higher in WARs E− (2.8±0.3g) than in WARs E+ (0.86±0.1g) or in Wistar (E− 1.4±0.02g or E+ 1.6±0.4g). After 10 min in zero-Ca2+, contraction induced by PHE was abolished in aorta from all groups, except in Wistar E− (0.6±0.01g). Ca2+addition in presence of PHE did not produce contraction in all groups. Thapsigargin (1μM-TG) in Ca2+-free medium did not produce contraction in any group. When Ca2+ solution was added, in presence of TG, only aorta from Wistar rats contracted (E− 0.5±0.01g; E+ 0.5±0.2g). Contraction induced by PHE in Ca2+ solution plus TG, did not recover only in WARs E+ (0.3±0.06g). In conclusion, the presence of endothelium prevents the contraction induced by PHE in aorta from acoustically stimulated WARs after SERCA inhibition. Demonstration that this response is endogenous to WARs will be investigated. Support: FAPESP, CNPq, PROAP-CAPES

EEG wavelet analyses of the striatum– substantia nigra pars reticulata– superior colliculus circuitry: Audiogenic seizures and anticonvulsant drug administration in Wistar audiogenic rats (War strain)

The importance of the substantia nigra pars reticulata (SNPr), striatum (STR) and superior colicullus (SC) in the blockade of experimental seizures is well known. But, in audiogenic seizures (brainstem tonic-clonic seizures), the anticonvulsant activity of these nuclei is still controversial. In the present study we aimed to analyze the STR–SNPr–CS circuitry in the audiogenic seizures of Wistar audiogenic rat (WAR). Behavioral and electroencephalographic (EEG) data were collected from WARs under no treatment or injection with systemic (phenobarbital) or intracerebral (intranigral) drugs (muscimol and phenobarbital). The main EEG frequency oscillation of STR, SNPr and SC seen before, during and after audiogenic seizures or during seizure protection, was determinated with wavelet spectral analyses. This method allows the association between behavior and EEG (video-EEG). Audiogenic seizures last only for half a minute in average, suggesting that the interruptions of seizures are probably not due to exhaustion. Systemic phenobarbital caused an acute and dose-dependent behavioral and EEGraphic anticonvulsant effect both in WARs. The dose of phenobarbital 15 mg/kg protected animals almost completely, without side effects such as ataxia and sedation. In our data, this endogenous “natural” seizure blockade (or termination) seems to be similar to the “forced” seizure abolition, like the one caused by a systemic non-ataxic phenobarbital dose, because in both cases an intense decrease in the EEG main frequency oscillation can be seen in SNPr and SC. Intranigral phenobarbital or muscimol did not protect animals, and actually induced an increase in the main EEG frequency oscillation in SC. The main finding of the present study is that, in contrast to what is well believed about the incapacity to control audiogenic seizures by the striato-nigro-tectal circuitry, we collected here evidences that these nuclei are involved in the ability to block these seizures. However, the striato-nigro-tectal circuitry in WARs, a genetically developed strain, seems to have different functional mechanisms when compared with normal rats.

Vasopressinergic hypothalamic neurons are recruited during the audiogenic seizure of WARs

The Wistar Audiogenic Rat (WAR) is a genetic model of reflex epilepsy with seizures induced by high-intensity sound stimulation (120 dB SPL). In spite of the known neural substrates involved in WAR seizure phenotype, neuroendocrine hypothalamic neurons were never investigated. In this work, AVP immunohistochemistry in the hypothalamus and radioimmunoassay (RIA) in plasma and in hypothalamic and hypophysial tissues were performed on both controls and WARs in order to evaluate the dynamics of AVP release due to seizure induction. Susceptible animals (WARs) displayed at least tonic–clonic convulsions followed by clonic spasms, while resistant Wistar rats (R) had no convulsive behavior. Animals were sacrificed at 3 instances: basal condition (without stimulus) and at 3 and 10 min after sound stimulation. For the immunohistochemistry AVP study, brains were harvested and processed by the avidin–biotin–peroxidase detection method. Optic densitometry was used for quantifying AVP labeling in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. SON presented higher densitometry levels (% D—relative to background) for both WARs and R when compared to PVN. Nevertheless, both nuclei presented a marked decrease, referenced to basal levels, in % D for WARs at 3 min (~35%) against a discrete change for R (~90%). RIA results were significantly higher in the hypophysis of WARs when compared to R rats, at 3 min. Also, at 3 min, plasma AVP in WARs (89.32 ± 24.81 pg/mL) were higher than in R (12.01 ± 2.39 pg/mL). We conclude, based on the AVP releasing profiles, that vasopressinergic hypothalamic neurons are recruited during the audiogenic seizure of WARs.

Electrophysiological properties of cultured hippocampal neurons from Wistar Audiogenic Rats

The main goal of this work was to analyze the electrophysiological properties of cultured hippocampal neurons from a particular epileptic rat strain, called Wistar Audiogenic Rats (WAR). The whole-cell patch-clamp technique was used to record both active and passive membrane responses in an attempt to detect alterations in their characteristics in relation to controls from Wistar rats. Neurons from WARs show a significant reduction in the magnitude of the inhibitory GABAergic currents (∼45%), in spite of maintaining a normal level of the excitatory glutamatergic currents. In addition, the magnitude of potassium currents, measured at +80 mV, is reduced by about 30% in comparison to controls. Surprisingly, we also found important changes in the passive cellular properties in WAR neurons such as membrane potential (−50.0 mV in WARs and −63.1 mV in controls) and input resistance (647 MΩ in WARs and 408 MΩ in controls). The changes described here, could be the basis of the neurophysiological and behavioral alterations present in these hyperexcitable animals, contributing to a better understanding of epileptogenesis in this particular animal model.

Evidence for augmented brainstem activated forebrain seizures in Wistar Audiogenic Rats subjected to transauricular electroshock

Previous work from our laboratory has shown that naïve Wistar Audiogenic Rats (WARs), a genetic model of reflex epilepsy in which seizures are induced by high-intensity sound stimulation (120 dB SPL), are seizure-prone to a variety of pro-convulsive stimuli (e.g., transauricular electroshock, pentylenetetrazole and pilocarpine). On the other hand, repetitive acoustic stimulation of WARs causes a slow recruitment of limbic structures, known as audiogenic kindling, changing seizure expression to include behavior characteristic of temporal-lobe epilepsy. Thus, our hypothesis is that WARs have facilitated acoustic-limbic projections when compared to Wistar controls. Wistar controls ( n = 9) and WARs ( n = 9) underwent EEG electrode implants in the cortex-Cx, amygdaloid complex-AMY and inferior colliculus-IC and received one low current transauricular electrical stimulus (ES) daily, for three consecutive days, with intensities of 10, 20 and 30 mA, respectively. The video-electroencephalographic activity was recorded 1 min before and 4 min after ES. Our results confirm previously described data indicating a greater susceptibility of WARs to seizure. However, low current ES (e.g., 20 mA) triggered epileptiform activity in the AMY only after epileptiform EEG was visible in the Cx and IC electrode leads. The AMY after-discharge continued even though no evident epileptiform activity was present in the Cx. In conclusion, our results add electrophysiological data to previously published behavioral evidence of WAR enhanced susceptibility to ES seizures and, also, support the hypothesis that the acoustic-limbic circuitry is facilitated even in unkindled WARs.

Neuroethological study of status epilepticus induced by systemic pilocarpine in Wistar audiogenic rats (WAR strain)

The administration of pilocarpine (PILO) is widely recognized as resulting in an experimental model of temporal lobe epilepsy; it is characterized by induction of status epilepticus (SE) and spontaneous recurrent seizures after a latent period. We provide in this work a neuroethological description of the SE induced by PILO. Behavioral evaluations were made in Wistar Audiogenic Rats (WARs) and Wistar resistant (R) animals. The experimental group (R) and WARs were pretreated with methyl scopolamine (1 mg/kg ip) and injected with PILO (R animals, 340–380 mg/kg ip; WARs, 240–280 mg/kg ip). Among R animals, 36% developed SE, and among WARs, 53%. The control group (R animals and WARs) was injected only with methyl scopolamine plus saline. The ETHOMATIC method was used for evaluation of seizures. Sequences included in the analysis were chosen using (1) fixed observation windows and (2) behavioral triggers. The R group showed that the threshold for seizure is variable, so seizure onset and behavioral evolution were better described using behavioral triggers than fixed observation windows. The observation windows selected in similar duration intervals do not characterize the seizures. Sequential analysis in the WAR group showed high mortality after SE and greater susceptibility to PILO, compared with R animals. We conclude that with neuroethological tools it is possible to better map the sequence and evolution of SE induced by PILO compared to only using behavioral and arbitrary seizure severity scales. This sequence is faster and stronger in severity when WARs are compared with R animals. Although the WARs underwent an evolution of SE in some way equivalent to that of R animals, some rats presented tonic–clonic convulsions after PILO injection, very similar to acute audiogenic seizures, a brainstem-dependent model. The current data also point to the PILO-plus-WAR combination as a suitable protocol to study the genetic–epilepsy connection in experimental temporal lobe epilepsy.

Limbic epileptogenicity, cell loss and axonal reorganization induced by audiogenic and amygdala kindling in wistar audiogenic rats (WAR strain)

Audiogenic seizures are a model of generalized tonic–clonic brainstem-generated seizures. Repeated induction of audiogenic seizures, in audiogenic kindling (AuK) protocols, generates limbic epileptogenic activity. The present work evaluated associations between permanence of AuK-induced limbic epileptogenicity and changes in cell number/gluzinergic terminal reorganization in limbic structures in Wistar audiogenic rats (WARs). Additionally, we evaluated histological changes after only amygdala kindling (AmK) and only AuK, and longevity of permanence of AuK-induced limbic epileptogenicity, up to 160 days. WARs and Wistar non-susceptible rats were submitted to AuK (80 stimuli) followed by both 50 days without acoustic stimulation and AmK (16 stimuli), only AmK and only AuK. Cell counting and gluzinergic terminal reorganization were assessed, respectively, by using Nissl and neo-Timm histochemistries, 24 h after the last AmK stimulus. Evaluation of behavioral response to a single acoustic stimulus after AuK and up to 160 days without acoustic stimulation was done in another group. AuK-induced limbic epileptogenicity developed in parallel with a decrease in brainstem-type seizure severity during AuK. AmK was facilitated after AuK. Permanence of AuK-induced limbic epileptogenicity was associated with cell loss only in the rostral lateral nucleus of amygdala. Roughly 20 generalized limbic seizures induced by AuK were neither associated with hippocampal cell loss nor mossy fiber sprouting (MFS). AmK developed with cell loss in hippocampal and amygdala nuclei but not MFS. Main changes of gluzinergic terminals after kindling protocols were observed in amygdala, perirhinal and piriform cortices. AuK and AuK-AmK induced a similar number and type of seizures, higher than in AmK. AmK and AuK-AmK were associated with broader cell loss than AuK. Data indicate that permanent AuK-induced limbic epileptogenicity is mainly associated to gluzinergic terminal reorganization in amygdala but not in the hippocampus and with no hippocampal cell loss. Few AmK-induced seizures are associated to broader and higher cell loss than a higher number of AuK-induced seizures.

Do endogenous opioids and nitric oxide participate in the anticonvulsant action of dipyrone?

It was previously reported that systemic administration of dipyrone inhibited the tonic component of generalized tonic-clonic seizures in both the electroshock and the audiogenic seizure models. The aim of the present study was to investigate the mechanisms involved in the anticonvulsant action of dipyrone by assessing the role of nitric oxide and opioids in the electroshock (female 60- to 90-day-old Wistar rats, N = 5-11) and audiogenic seizure (female 60- to 90-day-old Wistar audiogenic rats, N = 5-11) models of epilepsy. Naloxone (5 mg/kg, sc) significantly reversed the anticonvulsant effect of dipyrone in rats submitted to the induction of audiogenic seizures (ANOVA/Bonferroni's test), suggesting the involvement of opioid peptides in this action. In the electroshock model no reversal of the anticonvulsant effect of dipyrone by naloxone (5 mg/kg, sc) was demonstrable. The acute (120 mg/kg, ip) and chronic (25 mg/kg, ip, twice a day/4 days) administration of L-NOARG did not reverse the anticonvulsant action of dipyrone in the audiogenic seizure model, suggesting that the nitric oxide pathway does not participate in such effect. Indomethacin (10, 20 and 30 mg/kg, ip) used for comparison had no anticonvulsant effect in the audiogenic seizure model. In conclusion, opioid peptides but not nitric oxide seem to be involved in the anticonvulsant action of dipyrone in audiogenic seizures.