Mapping of c-Fos expression in the rat brain during the evolution of pentylenetetrazol-kindled seizures

c-Fos protein immunocytochemistry was used to map the brain structures recruited during the evolution of seizures that follows repeated administration of a subconvulsive dose (35 mg/kg, ip) of pentylenetetrazol in rats. c-Fos appeared earliest in nucleus accumbens shell, piriform cortex, prefrontal cortex, and striatum (stages 1 and 2 of kindling in comparison to control, saline-treated animals). At the third stage of kindling, central amygdala nuclei, entorhinal cortex, and lateral septal nuclei had enhanced concentrations of c-Fos. At the fourth stage of kindling, c-Fos expression was increased in basolateral amygdala and CA1 area of the hippocampus. Finally, c-Fos labeling was enhanced in the dentate gyrus of the hippocampus only when tonic–clonic convulsions were fully developed. The most potent changes in c-Fos were observed in dentate gyrus, piriform cortex, CA1, lateral septal nuclei, basolateral amygdala, central amygdala nuclei, and prefrontal cortex. Piriform cortex, entorhinal cortex, prefrontal cortex, lateral septal nuclei, and CA3 area of the hippocampus appeared to be the brain structures selectively involved in the process of chemically induced kindling of seizures.     

Decreased expression of brain cAMP response element-binding protein gene following pentylenetetrazol seizure

The present study investigated whether the expression of the cAMP response element-binding protein (CREB) in the rat brain is altered following an acute self-limited seizure induced by pentylenetetrazol (PTZ). Male rats were injected intraperitoneally with a single convulsive dose (45 mg/kg) of PTZ, and the matched controls were given saline. For immunohistochemistry, animals were perfused with 4% parafomaldehyde at 24 h following PTZ seizures, and CREB immunoreactivity was examined in rat brain. For real-time RT-PCR, animals were sacrificed at 2 and 24 h and 1 week following PTZ seizures. Tissues from different rat brain regions were micropunched and subjected to real-time RT-PCR using Taqman probe. The CREB immunoreactive profiles were significantly decreased in CA3 and dentate gyrus of hippocampal formation, sensory cerebral cortex and thalamus at 24 h after PTZ seizures. Consistent with changes in CREB immunoreactivity, levels of CREB mRNA were significantly decreased in the hippocampus, cerebral cortex, amygdala and thalamus at 24 h after PTZ seizures. No significant change was found for CREB mRNA expression in these regions at 2 h or 1 week following PTZ seizures. These results show that a brief seizure caused a decline in CREB expression up to 24 h later.     

Neuropathological profile of the pentylenetetrazol (PTZ) kindling model

ABSTRACT

Introduction: There are three phases of seizure developing in pentylenetetrazol (PTZ)-induced kindling animal model: (i) pre-kindling phase; (ii) kindling phase or after animals are fully kindled; (iii) post-kindling phase with non-provoked spontaneous recurrent seizures. The aims of this review were to summarize the progress over time of the electroencephalographic features and neuropathological alterations in kindled PTZ treated animals.

Materials and methods: Keywords relevant to PTZ kindling were used to a guide a literature search on Pubmed, Medline and Cochrane Library.

Results: Clonic seizures induced PTZ at kindling phase led to a strong c-Fos expression in the hippocampus. Although, decline hippocampal neuron and metabolism disturbances were detected at pre-kindlig phase. Repeated PTZ induced seizures alter the GABA-mediated inhibition and glutamate-mediated excitation, which may contribute to increased seizure susceptibility. Similar to chemical animal models such as the pilocarpine and the kainic acid models, mossy fiber sprouting, hippocampal damage, and glucose hypometabolism had been seen after PTZ induced seizures.

Conclusion: PTZ kindling model may improve understanding of the seizures development provided that the differences existing between the phases of kindling model are taken into account.     

Differences in c-fos immunoreactivity due to age and mode of seizure induction

The aim of this study was to determine whether the regional distribition and time course of immunoreactivity to the c-fos protein varies with maturation and method of seizure induction. The effect of the two chemical convulsants, pentylenetetrazol (PTZ) and flurothyl, on the spatial and temporal pattern of c-fos-like immunoreactivity in immature (postnatal day (P) 10) was compared to that in adult rats. Patterns of c-fos-like immunoreactivity following O₂ deprivation were also evaluated at the 2 ages because hypoxia is acutely epileptogenic in immature animals but not adults. C-fos-like immunoreactivity was examined at 2, 4, and 6 h after onset of chemically induced seizures or O₂ deprivation at both ages. After PTZ or flurothyl seizures, both ages exhibited similar patterns of IR in amygdala, pyriform cortex, and hypothalamus. Age-dependent regional differences were most prominent in cortex: superficial layers of retrosplenial, cingulate, and neocortex stained in adults; staining was confined to deep layers of neocortex in P10 rats. Intense staining of dentate gyrus and hippocampus occurred with more prolonged seizures, but not brief seizures. PTZ administration resulted in staining at 2 h after seizure onset and was reduced by 4 h in adults, but immunoreactivity was not seen until 4 and 6 h after seizure onset in immature rats, indicating an age effect on the time course of IR. In immature rats, immunoreactivity patterns after hypoxia were markedly different from PTZ or flurothyl; staining was confined to layer VI of neocortex in these animals, and rarely involved limbic structures. These differences in the pattern of c-fos immunoreactivity suggest that the neuronal populations involved in epileptogenesis are influenced by age as well as seizure phenotype and intensity.     

Development of long-lasting potentiation effects in the dentate gyrus during pentylenetetrazol kindling

In the present study kindling was induced in rats by repeated intraperitoneal injection of pentylenetetrazol (PTZ) once every 48 h. The resulting seizure stages were registered after each PTZ application. The development of PTZ-induced kindling and the time course of possible potentiation effects in the dentate gyrus were examined. The efficacy of perforant pathway transmission to the granule cells was tested in every second kindling session by measuring the monosynaptic evoked field potentials recorded in the dentate gyrus following single test stimuli of the perforant pathway at different times after PTZ injection in freely moving animals. The data suggest that establishment of a PTZ kindling is associated with the development of long-lasting potentiation of the field potentials. After completion of kindling it was demonstrated that kindled rats also show a diminished learning performance. The relationship between the development of potentiation phenomena in hippocampal substructures and learning impairment is discussed.     

戊四氮点燃致大鼠癫痫状态后脑内HSP70的分布研究

目的：了解热休克蛋白70（HSP70）在癫痫状态大鼠脑内的分布。方法：应用免疫细胞化学方法观察戊四氮（PTZ）点燃致大鼠癫痫状态后脑内不同区域HSP70的表达。结果：PTZ点燃后大鼠脑CA3区、梨状皮层、杏仁核、丘脑、内嗅皮层、CA1区、顶叶皮层HSP70表达依次减少。结论：HSP70在脑内的表达分布与癫痫脑神经元对兴奋性损伤的耐受性密切相关,可能是癫痫脑神经元选择性脱失的内在机制之一。     

Antiepileptic activity of melatonin in guinea pigs with pentylenetetrazol-induced seizures

Abstract

Background: Antiepileptic and neuroprotective effects of melatonin (N-acetyl-5-methoxytryptamine) have been shown at higher doses (50–160 mg/kg). We aimed to investigate the antiepileptic effects of low-dose melatonin (10 mg/kg) on pentylenetetrazol (PTZ)-induced experimental epilepsy model.

Materials and Methods: Twelve male albino guinea pigs weighing 500–800 g were used in our work. Initially, latent period, seizure intensity and mortality parameters were evaluated during the epileptic seizure induced by PTZ. After a recovery period of 7 days, effects of the neuroprotective agent, melatonin (which is dissolved in 2.5% ethanol–saline solution), on epileptic seizures induced by PTZ were evaluated. Effects of 2.5% ethanol, which is an anticonvulsant agent when administered acutely in high concentrations, on PTZ-induced seizures were also evaluated.

Results: Data obtained from the study groups (PTZ, PTZ + melatonin and PTZ + ethanol) were evaluated by paired t-test, and p<0.005 was considered statistically significant. The differences of latent periods between the PTZ and PTZ + melatonin groups were found to be statistically significant (p<0.001).

Conclusion: Although melatonin does not have a primary anticonvulsant effect at low doses (10 mg/kg), it lowers the mortality rates and attenuates seizure severity while increasing the latent period.     

Effect of minocycline on pentylenetetrazol-induced chemical kindled seizures in mice

Inflammation is one of the mechanisms involved in seizure induction. In this study, the effect of minocycline, an anti-inflammatory drug, was investigated on kindling acquisition. Chemical kindling was induced by injection of a subthreshold dose of pentylenetetrazol (PTZ; 37.5 mg/kg) in mice on every other day. Two groups of animals received minocycline (25 mg/kg) at 1 h before or 1 h after PTZ injection. Following the last PTZ injection, the changes in gene expression of TNF-α receptor, γ₂ subunit of GABA_A receptor and NR₂A subunit of NMDA receptor were assessed in the hippocampus and piriform cortex. Injection of minocycline before PTZ increased the latency to stage 4 seizure, and decreased the duration of stages 4 and 5 seizure. It also prevented the increase in the mRNA of NR₂A subunit of NMDA receptor in the hippocampus and removed the PTZ-induced increase in mRNA of γ₂ subunit of GABA_A receptor in piriform cortex of PTZ kindled mice. Minocycline also prevented the increase in TNF-α receptor gene expression in both hippocampus and piriform cortex. Injection of minocycline after PTZ had no significant effect on measured parameters. Therefore, it can be concluded that minocycline may exert an anticonvulsant effect through preventing the increase in GABA_A and NMDA receptor subunits. These effects are accompanied by a reduction in an important inflammation index, TNF-α receptor.     

D-Galactose injured neurogenesis in the hippocampus of adult mice

Abstract

Objectives: We studied the effects of the reactive oxygen species (ROS) on neural progenitor cell proliferation and survival in the dentate gyrus (DG).

Methods: The adult mice were treated with D-galactose for 7 weeks to mimic natural aging in mice. The level of malondialdehyde (MDA) and the activities of antioxidant enzymes in the serum were detected. Neurodegeneration and neurogenesis in the hippocampus were explored using terminal deoxynucleotidyltransferase-mediated UTP nick-end labeling (TUNEL) to detect the dying cells and bromodeoxyuridine (BrdU) was used to label the newly born cells.

Results: After the treatment of D-galactose, the level of MDA increased and the activities of the antioxidant enzyme decreased in the serum. TUNEL-positive cells significantly increased in the DG, CA1 and CA3 subfields. The BrdU-labeled proliferating cells and surviving cells in the DG decreased significantly in number after D-galactose treatment.

Discussion: D-Galactose induced the impairment of neurogenesis in the DG, which is similar to natural aging in mice. ROS accumulation as a result of D-galactose may be related to the decrease of neurogenesis in the DG.     

Anterior thalamic mediation of generalized pentylenetetrazol seizures

The effects of microinjection of various neuroactive compounds into the anterior thalamic nucleus (AN) and other selected subcortical regions of guinea pig brain on the expression of pentylenetetrazol (PTZ)-induced behavioral and electrical seizure activity were examined. Excitatory agents, kainic acid (KA), bicuculline (BIC) or PTZ, injected into the AN or other thalamic nuclei, striatum, but not the mammillary bodies (MB), facilitated the EEG convulsant action of systemically administered PTZ. Injection of muscimol into the AN protected against the expression of PTZ-induced repetitive high-voltage EEG seizure discharges and inhibited the facilitatory effects of subcortically applied KA or BIC. Injection of muscimol into the AN was also able to terminate established ongoing seizure discharges. Unilateral application of muscimol to the AN did not prevent the repetitive hypersynchronous EEG discharges following systemic PTZ but did result in the delay in the onset of cortical hypersynchrony in the ipsilateral hemisphere. Muscimol injections into other thalamic nuclei, MB, cortex, striatum or directly into the CSF space had no anticonvulsant effect, however. Microinjection of gamma-vinyl-gamma-aminobutyric acid, a selective GABA transaminase inhibitor, resulted in protection from the behavioral convulsant action and lethal effects of PTZ when administered into the thalamus, especially the AN, but not when injected into the striatum or CSF. These data demonstrate that the AN is an important subcortical nucleus for the mediation of both cortical EEG synchrony and behavioral seizure expression induced by PTZ. In light of previous results establishing a role for the brainstem and diencephalon in PTZ seizure expression, the AN may serve, in part, as a gating mechanism for the propagation of paroxysmal activity between subcortical areas and the cerebral cortex.     

不同化学致癇剂对大鼠癫癇发作和神经元凋亡的影响

目的观察几种不同癫癇持续状态模型发作的特点和海马区的组织病理学改变.方法采用匹罗卡品、锂-匹罗卡品和戊四氮腹腔注射制成大鼠癫持续状态模型,以TUNEL方法标记DNA片段,原位检测海马CA1和CA3区的凋亡神经细胞.结果海马CA1和CA3区神经细胞凋亡数,PILO组多于Li-PILO组和PTZ组,差异有显著性(P《0.05).结论匹罗卡品、锂-匹罗卡品和戊四氮均可诱发大鼠癫持续状态,并导致海马神经元损伤.     

戊四氮致痫大鼠脑内热休克蛋白70的表达

目的：探讨热休克蛋白70（HSP70）在癫痫大鼠脑内的表达情况及其意义。方法：采用戊四氮致痫模型。应用免疫组织化学及常规清理检查的方法进行研究。结果：在正常大鼠脑内未见HSP70免疫反应（IR）阳性细胞,成四氮致痫12小时后脑内开始出现HSP70IR阳性细胞,24小对IR达高峰．3天后开始下降．7天后消失。HSP70主要在边缘系统（尤其是海马的CA1、CA3、CA4区）、大脑皮质（尤其是颞叶皮质,梨状皮质）等区域表达。同时常规病理检查发现,上述区域散在出现受损的异常神经元。结论：癫痫发作可诱导HSP70在大鼠脑内广泛表达。HSP70表达可作为神经元受损的一个早期指标。     

BOLD-fMRI of PTZ-induced seizures in rats

PURPOSE: To develop a non-invasive method for exploring seizure initiation and propagation in the brain of intact experimental animals. METHODS: We have developed and applied a model-independent statistical method--Hierarchical Cluster Analysis (HCA)--for analyzing BOLD-fMRI data following administration of pentylenetetrazol (PTZ) to intact rats. HCA clusters voxels into groups that share similar time courses and magnitudes of signal change, without any assumptions about when and/or where the seizure begins. RESULTS: Epileptiform spiking activity was monitored by EEG (outside the magnet) following intravenous PTZ (IV-PTZ; n=4) or intraperitoneal PTZ administration (IP-PTZ; n=5). Onset of cortical spiking first occurred at 29+/-16 s (IV-PTZ) and 147+/-29 s (IP-PTZ) following drug delivery. HCA of fMRI data following IV-PTZ (n=4) demonstrated a single dominant cluster, involving the majority of the brain and first activating at 27+/-23s. In contrast, IP-PTZ produced multiple, relatively small, clusters with heterogeneous time courses that varied markedly across animals (n=5); activation of the first cluster (involving cortex) occurred at 130+/-59 s. With both routes of PTZ administration, the timing of the fMRI signal increase correlated with onset of EEG spiking. CONCLUSIONS: These experiments demonstrate that fMRI activity associated with seizure activity can be analyzed with a model-independent statistical method. HCA indicated that seizure initiation in the IV- and IP-PTZ models involves multiple regions of sensitivity that vary with route of drug administration and that show significant variability across animal subjects. Even given this heterogeneity, fMRI shows clear differences that are not apparent with typical EEG monitoring procedures, in the activation patterns between IV and IP-PTZ models. These results suggest that fMRI can be used to assess different models and patterns of seizure activation.     

Imaging the neural substrates involved in the genesis of pentylenetetrazol-induced seizures

PURPOSE: Functional imaging of animal models makes it possible to map the functional neuroanatomy contributing to the genesis of seizures. Pentylenetetrazol (PTZ)-induced seizure in rats, a relevant model of human absence and of generalized tonic-clonic epilepsy, was used to stimulate seizure activity within 30 s of administration while collecting continuous, high-resolution, multislice images at subsecond intervals. METHODS: Pilot studies were conducted to establish a quick and effective PTZ model for the imaging experiments. PTZ was then used to stimulate seizure activity in rats while collecting multislice functional MRI (fMRI) images from the entire forebrain at 4.7 Tesla. Ethosuximide (ESM) also was used to block seizure activity. RESULTS: Within 2-4 s of PTZ administration, a rapid increase in blood oxygen level-dependent (BOLD) signal intensity was noted in the thalamus, especially the anterior thalamic nuclei. Activity in the anterior thalamus peaked approximately 15 s before seizure onset and was more than twofold greater than that in all other thalamic areas. The retrosplenial cortex showed a twofold greater increase in activity as compared with other cortical areas, also peaking at approximately 15 s. The dentate gyrus was twice as active as other hippocampal areas but peaked just before seizure onset. Treatment with ESM blocked seizures, decreasing PTZ-induced activation in most forebrain areas. The anterior thalamus and retrosplenial cortex were essentially blocked by pretreatment with ESM. CONCLUSIONS: The anterior thalamus, retrosplenial cortex, and dentate gyrus show the greatest increases in BOLD signal activity before seizure onset. Neurons in these areas may contribute to the neural network controlling the initiation of generalized tonic-clonic seizure.     

Immunohistochemical analysis of ubiquilin‐1 in the human hippocampus: Association with neurofibrillary tangle pathology

This post mortem immunohistochemical study examined the localization and distribution of ubiquilin‐1 (UBL), a shuttle protein which interacts with ubiquitin and the proteasome, in the hippocampus from Alzheimer's disease (AD) dementia cases, and age‐matched cases without dementia. In Braak stages 0–I–II cases, UBL immunoreactivity was detected in a dense fiber network in the neuropil, and in the cell cytoplasm and nucleoplasm of neurons in Cornu Ammonis (CA) fields and dentate gyrus granular neurons. In Braak stages III‐IV and V‐VI cases, UBL immunoreactivity was reduced in the neuropil and in the cytoplasm of the majority of CA1 neurons; some CA1 pyramidal neurons and the majority of CA2/3 pyramidal, CA4 multipolar, and dentate granular neurons had markedly increased UBL immunoreactivity in the nucleoplasm. Dual immunofluorescence analysis of UBL and antibody clone AT8 revealed co‐localization most frequently in CA1 pyramidal neurons in Braak stage III‐IV and V‐VI cases. Further processing using the pan‐amyloid marker X‐34 revealed prominent UBL/X‐34 dual labeling of extracellular NFT confined to the CA1/subiculum in Braak stage V‐VI cases. Our results demonstrate that in AD hippocampus, early NFT changes are associated with neuronal up‐regulation of UBL in nucleoplasm, or its translocation from the cytoplasm to the nucleus. The perseverance of UBL changes in CA2/3, CA4 and dentate gyrus, generally considered as more resistant to NFT pathology, but not in the CA1, may mark a compensatory, potentially protective response to increased tau phosphorylation in hippocampal neurons; the failure of such a response may contribute to neuronal degeneration in end‐stage AD.     

Temporal Rearrangement of Pre-ictal PTZ Induced Spike Discharges by Low Frequency Electrical Stimulation to the Amygdaloid Complex

BackgroundEpilepsy is a common neurological disease affecting over 40 million people worldwide. The foremost important challenge of epileptologists has been to control and predict the recurrent and spontaneous seizures of epileptic patients. The application of low frequency electrical stimulation (LFS) in deep brain structures has shown promising results in seizure control. However, the use of LFS as a probing strategy for seizure prediction, thus contributing to a closed loop solution, is still poorly explored.ObjectiveTo improve seizure prediction by producing gradually increasing phase-locked pre-ictal electrographical responses, due to the short-term plastic changes in epileptogenic neural networks, thus behaving as a “programmed” surrogate marker.MethodsUrethane anesthetized rats were divided into 3 groups: the PTZ-noES group was injected with pentylenetetrazole (PTZ 4 mg/ml/min flow rate) i.v. without electrical stimulation (ES); the ES-noPTZ group received ES (0.5 Hz, 0.1 ms pulse width and 0.6 mA) to the amygdaloid complex and the PTZ + ES group received simultaneously i.v. PTZ infusion and ES. After each condition, electrographical parameters and c-Fos expression of regions of interest were evaluated.ResultsAlthough the PTZ + ES group had no evident change in the sustained electrographic seizure onset, duration and/or frequency spectrum; c-Fos labeling showed a different expression pattern when compared to the PTZ-noES and ES-noPTZ. Also, PTZ + ES formed a gradually increasing evoked potential; confirming the strong coupling of reverberant neural networks induced by ES – phase locked to stimuli.ConclusionES induces a detectable temporal rearrangement of pre-ictal activity, which has suggestive applicability to seizure prediction.     

Mapping of neuronal networks underlying generalized seizures induced by increasing doses of pentylenetetrazol in the immature and adult rat: a c‐Fos immunohistochemical study

Previous studies from our group have shown that pentylenetetrazol (PTZ)-induced status epilepticus (SE) leads to age-dependent acute and long-term metabolic and circulatory changes in immature rats. In order to define the neural substrates involved in PTZ seizures according to age, the purpose of the present study was to map the areas of cellular activation during seizures of increasing severity in 10-day-old (P10), 21-day-old (P21) and adult rats. Seizures were induced by repetitive injections of subconvulsive doses of PTZ. The total dose received by the animals ranged from 4 to 125 mg/kg. These doses induced a variety of seizure profiles including absence-like, clonic seizures and SE. The cellular activation was measured as the density of c-Fos immunoreactive cells in animals at 2 h after the onset of the seizures. In P10 rats receiving a behaviourally non-active dose of PTZ, c-Fos immunoreactivity appeared only in the amygdala. The dose of 40 mg/kg that induced absence-like seizures led to a weak c-Fos expression in the medial thalamus, some cortical areas and globus pallidus. Clonic seizures reinforced labelling in the previous areas and induced a spread of c-Fos immunoreactivity to other cortical areas, thalamus, hypothalamus and some brainstem nuclei. At that age, only SE led to a widespread and stronger expression of c-Fos which was, however, totally lacking in the midbrain, and remained incomplete in the brainstem and forebrain limbic system, including the hippocampus. In P21 and adult rats, the inactive dose of PTZ induced c-Fos immunoreactivity in thalamus and hypothalamus. With absence-like seizures, c-Fos labelling spread to the cerebral cortex, amygdala, septum and some brainstem regions. With clonic seizures, immunoreactivity was reinforced in all areas already activated by absence-like seizures, and appeared in the striatum, accumbens, brainstem and hippocampus, except in CA1. After SE, c-Fos was strongly expressed in all brain areas. The intensity of c-Fos labelling was higher in most regions of P21 compared to adult rats. These data are in agreement with the immaturity of cellular and synaptic connectivity in P10 rats, the known greater sensitivity of rats to various kinds of seizures during the third week of life and the nature of the neural substrates involved in PTZ seizures.     

Effect of seizures kindled by subconvulsant doses of pentylenetetrazol on dopamine receptor binding and dopamine-sensitive adenylate cyclase in the rat

Possible alterations in dopamine receptor regulation were assessed in pentylenetetrazol (PTZ)-kindled rats by examination of dopamine (DA)-sensitive adenylate cyclase activity and the specific binding of [³H]spiroperidol in various brain regions. A reduction in [³H]spiroperidol-labeled high-affinity binding sites in the amygdaloid-pyriform cortex followed the kindling of seizures by chronic administration of PTZ. Similarly, a reduction of low-affinity binding sites was observed in the frontal cortex of PTZ-kindled rats. No changes in the apparent affinity of both high- and low-affinity binding sites were found after kindling except in the amygdala-pyriform cortex. The affinity of the low-affinity binding site was significantly reduced in this brain region. The lack of significant changes in the number and affinity of binding sites after acute seizures induced by electroconvulsive shock or a convulsant dose of PTZ suggests that the changes in receptor binding after PTZ kindling were not related to seizure sequelae. Basal and DA-sensitive adenylate cyclase activities remained unchanged after PTZ kindling. In conjunction with our previous observations of significant alterations in [³H]spiroperidol binding after amygdaloid kindling, the present findings suggest that altered DA receptor regulation may be a general mechanism involved in the development of increased seizure susceptibility.