| BackgroundTemporal lobe epilepsy (TLE) is the most common type of epilepsy among adult patients, accounting for about 65% of with focal epilepsy. Nearly one third of TLE patients do not respond well to currently available anti-epileptic medications. In addition to the presence of recurrent seizures, patients with TLE frequently display other comorbids such as cognitive dysfunction, depression and other social adaptive behaviors. Multiple histopathological changes such as loss of neurons, gliosis, synaptic reorganization, granule cell dispersion, and altered neurogenesis are present in the hippocampal dentate gyrus. Because of these changes, the dentate gyrus has been studied extensively for its roles in the development of epilepsy and propagation of epileptic waves.Decrease in GABAergic inhibition has been considered to be the most important mechanism underlying epileptogenesis. In addition to excitatory granule cells and mossy cells, the dentate gyrus also contains GABAergic interneurons, which not only function to prevent runaway excitation and to control the temporal and spatial dynamics of granule cell firing, but also contribute to pattern separation of dentate gyrus. Unlike excitatory neurons in the same region, GABAergic interneurons in the dentate gyrus are diverse, varying in morphological appearances, neurochemical identities, electrophysiological properties, and axonal projection domains. Epilepsy is associated with numerous alterations in hippocampal dentate GABAergic interneurons. In both animal epilepsy models and humans with spontaneous seizures, certain subtypes of dentate GABAergic interneurons are reduced. Accordingly, GABAergic neurotransmission is altered. Reduction in the number of GABAergic cells and GABAergic neurotransmission are in line with that decrease of GABAergic inhibition contribute to the onset and development of epilepsy. In contrast to the traditional view that GABAergic dysfunction leads to epileptogenesis, accumulating experimental evidence suggests that activation of GABAergic interneurons is needed for generation of seizures.Genes expressed that do not require protein synthesis are termed as immediate early genes (IEGs). Expression of IEGs either at mRNA and/or at protein levels in turn can be used to profile nerve cell activity. c-fos, activity-regulated cytoskeleton associated protein (Arc or Arg3.1) have been reported to be up-regulated following seizures induced either by electroconvulsive stimuli or through administration of chemoconvulsants.The present study underwent to profile the activity status of GABAergic interneurons of hippocampal dentate gyrus through assessing expression of Arc and c-fos and recording spontaneous firing at near resting membrane potential in a rat pilocarpine model of epilepsy.Materials and MethodsPilocarpine-induced SE in rats Experiments were performed on Sprague-Dawley rats (160-180 g,6-7 weeks old). SE lasting for at least for 120 minutes was induced by intraperitoneally injected pilocarpine hydrochloride. Thirty minutes after animals were pretreated with an intraperitoneal injection of methylscopolamine and terbutaline (2 mg/kg body weight each),150 mg/kg of pilocarpine was injected. Another dose of pilocarpine (300 mg/kg) was given 20 minutes after the first dose. Following the same pretreatment schedule, the controls were given equivalent volume of saline twice at times identical to the pilocarpine-treated animals. The intensity of seizures was graded according to Racine stages. Animals acted as Racine stage Ⅱ or higher were kept.Transcardiac perfusionAnimals were sacrificed at four different points:1 hr,1 week,2 weeks or more than 10 weeks after SE. Each animal was deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (80 mg/kg). After complete paralysis, the animal was perfused through the left ventricle with heparinized saline, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) over a period of 40 minutes. The brain was removed from the skull upon completion of transcardial perfusion. Tissue block made by trimming off the cerebellum was post-fixed in the same fixative at 4℃ overnight.Tissue preparationThereafter, the tissue block was sequentially immersed in 10% sucrose in 0.1 M PBS for 4 hours,15% sucrose in 0.1 M PBS for 8 hours, and finally 20% sucrose in 0.1 M PBS at 4℃ overnight. After the tissue block was firmly embedded with chicken egg albumin-gelatin-glutaraldehyde medium, it was horizontally cut through the hippocampus into 40 μm-thick-sections with a vibratome to yield serial hippocampal sections. Hippocampal sections with "C" shaped dentate gyri from the ventral part of the hippocampal formation were orderly collected into 0.1 M PBS and used for detecting the expression of IEGs in GABAergic cells in the above mentioned 4 time points and the activation of different subtypes of interneurons more than 10 weeks after SE.Timm’stainingTimm’s staining was performed to evaluate the severity. After transcardiac perfusion, the brains were fixed in 4% paraformaldehyde at 4℃ overnight. Then they were transferred in a 30% sucrose solution for dehydration and protection. For histology, after separated and frozen,30-μm-thick hippocampal tissue was sectioned by a freezing microtome. We chose one in every six sections from the ventral part of the hippocampal formation. These sections were attached on coated glass slides, then placed in the section box to dry. The sections were developed in the dark for 45 min in a Timm’staining solution at 26 ℃. After washing, the slices were dehydrated in graded alcohol, cleared in xylene, and covered with neutral balsam.Double immunofluorescenceAfter sections were washed with 1XPBS, they were incubated with a blocking solution for 2.5 hrs at 4 ℃ to minimize non-specific stains. After blocking, sections were incubated with a primary antibody pair that was diluted with the blocking solution at 4℃ overnight. Thereafter, the sections were washed with 1XPBS for three times (15 minutes each), then transferred to secondary antibodies. The sections were incubated in the secondary antibody mixture for 150 min and then washed with 1XPBS. The primary and secondary antibody used is given in Materials and Methods Table 1.3.Confocal image acquisition and analysesAll stained sections were scanned with a Carl Zeiss Laser Scanning Microscope. After the dentate gyrus of each section was scanned under a 10X objective with a zoom 0.8, areas were randomly chosen to undergo scanning under a 20X objective (zoom 1). All images were taken under the same scanning parameter sets for individual immunoreactions. Positive cells were counted under Photoshop by darkening hilar background stains. The activation of GABAergic interneurons was determined by the number of GABAergic cells positive for c-fos/the total number of GABA+cells X 100% whereas activations of different subtypes were calculated by the number of marker-expressing cells positive for c-fos/the total number of marker-expressing cells X 100%.Acute brain section preparationAnimals were decapitated under deep ether anesthesia at least 10 weeks after SE onset or saline injection. The brain was removed to ice-cold high-Mg2+ artificial cerebrospinal fluid (ACSF) with 95% O2/5% CO2. Horizontal hippocampal slices (410 μm in thickness) were cut under a Leica VT1200S vibrating microtome. After cutting, the slices were incubated at 34.5℃ for 30 minutes to promote metabolic recovery.ElectrophysiologyA slice transferred into a submersion-type chamber was immobilized by a platinum ring and continuously perfused with 95% 02/5% CO2-saturated artificial cerebrospinal fluid. Interneuron-like cells along the border of the hilus and granule cell layer were visualized under a 60X water-immersion objective. After a tight seal (>2 GQ) was made, spontaneous currents were recorded for 5 min in cell-attached mode at a holding potential of-60 mV. Thereafter, the membrane was ruptured to make whole-cell recording. The resting membrane potential was determined immediately after break-in. Membrane time constant, input resistance, and membrane capacitance were calculated from the current response to a 10 mV hyperpolarizing voltage from the resting Vm applied for 200 ms. Evoked firings in response to stepped current injection were recorded in a current-clamped mode.Morphological development of biocytin-filled cellsAfter electrophysiological recordings, the electrode tip was gently withdrawn, and the slice was fixed with 4% paraformaldehyde in 1 XPBS at 4℃ overnight. Thereafter, the slice was cut into 60-μm-thick sections with a vibratome. The yielded sections were orderly collected into 1XPBS. The sections were then incubated in 30%(vol/vol) methanol/2%(vol/vol) H2O2 for 90 min to inactivate endogenous peroxidases. After washing with 1XPBS, sections were incubated in an avidin-horseradish peroxidase solution at 4℃ overnight. After washing again with 1XPBS, color was developed with diaminobenzidine/H2O2/nickel ammonium sulfate for 6 min. The sections was moved to double distilled water to stop the reaction. After rinsing, sections were mounted on glass slides, drying overnight. The next day, the slices were dehydrated in graded alcohol, cleared in xylene, and covered with neutral balsam.Data collection and statistical analysisQuantitative data are expressed as mean ± standard error of the mean (SEM). Before subjecting to statistical comparisons, Kolmogorov-Smirnov test was conducted to confirm or to reject the normality of data sets. Data were statistically compared by Dunnett’s post-hoc test following one-way analysis of variance (ANOVA), Bonferroni post hoc test following two-way ANOVA, unpaired t-test, or the Mann-Whitney test (the Wilcoxon rank sum test), as appropriate. P values less than or equal to 0.05 are considered to be statistically significant.Results1. Mossy fiber sprouting after pilocarpine-induced status epilepticus (SE)In the controls (Control), stained mossy fibers in black are mainly distributed in the hilus and the striatum lucidum of CA3 region with very faint stain to be seen in the granular cell layer. Ten weeks after the induction of SE, in addition to the hilus and striatum lucidum of CA3 region, mossy fibers are also present in granular cell (GC) layer and the inner molecular layer; they are termed as recurrent mossy fibers.2. Arc and c-fos are co-expressed in the same cells with parallel intensity but different subcellular localizations in both the resting and stimulated conditionsIn the controls sacrificed 1 hour or more than 10 weeks after saline injection, both c-fos and Arc were found to be expressed in the dentate gyrus at lower intensity in a cell type-dependent manner. Arc and c-fos were both up-regulated 1 hour and more than 10 weeks after SE but the patterns of up-regulation at the two time points were quite different. One hour after SE, cells with the highest intensity of Arc or c-fos were present in the granule cell layer, whereas cells expressing Arc or c-fos with highest density more than 10 weeks after SE were present in the hilus.3. A biphasic increase in the percentage of hilar GABAergic cells positive for c-fos after pilocarpine-induced SEUsing controls and SE groups sacrificed at 1 hour,1 week,2 weeks and more than 10 weeks, we colocalizing c-fos with two GABAergic markers GAD67 and GABA, respectively. The results showed the expression characteristics of c-fos in GABA positive neurons were similar to the GAD67 positive cells, but c-fos expression in GABA was stronger. In the controls at any time point, c-fos expression in GABA-positive cells was low and no significant differences were revealed among those controls sacrificed at each time point. One hour after SE, up-regulation of c-fos was found not only in granule cells but also in GABA-positive cells. Although there was a trend of c-fos up-regulation, data observed 1 week after SE were not significantly different from those of controls. Increased c-fos expression in GABAergic cells reemerged 2 weeks after SE. More than 10 weeks after SE, c-fos expression in GABAergic interneurons became more frequent.4. Up-regulation of c-fos in GABAergic interneurons is subtype-dependentBecause a large portion of GABAergic cells expressed Arc and c-fos densely more than 10 weeks after SE, we attempted to determine if neurochemically defined GABAergic cells expressed IEGs in a subtype-dependent manner. To this end, c-fos was co-labeled with individual interneuron markers. In the controls, percentage of c-fos expression in neurochemically defined subtypes ranged from the highest 27.1% in PV-positive cells to the lowest 14.9% in CR-positive cells. More than 10 weeks after SE, with the exception of CR-positive cells, the percentage of GABAergic cells positive for c-fos significantly increased in all subtypes of GABAergic interneurons. Specifically, the percentage differences between the controls and SEs in CR-, nNOS-. NPY-, PV-, and SOM-positive subtypes are 9.9%,16.3%,46.2%,19.0% and 30.3%, respectively.5. Most hilar interneurons fire spontaneously more than 10 weeks after SEMorphologies developed from biocytin-filling cells showed that the control group included one basket, three TML, three HICAP and two HIPP cells whereas the SE group had one basket, three TML, two HICAP and three HIPP cells in which only one HIPP cell did not fire spontaneously. Two out of nine interneurons recorded from the controls fired spontaneously in cell-attached mode. In contrast, eight out of the nine interneurons recorded more than 10 weeks after SE fired spontaneously. The spontaneous firing frequency at -60 mV was significantly higher in SE group than that of the control group. Through statistical analysis, there were no significant differences in the levels of resting membrane potentials and the threshold potentials between Controls and SEs.Conclusions1. After a silent period after pilocarpine-induced status epilepticus, hilar GABAergic interneurons become hyperactive.2. The hyperactivity of hilar GAB Aergic interneurons vary among neurochemically defined interneuron subtypes.3. A persistent hyperactivity of GAB Aergic cells after SE may reflect a fragile balance between excitation and inhibition in the dentate network; this could serve as a mechanism in contributing to epileptiform activity. |
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