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It would be useful to selectively block granule cell axon (mossy fiber) sprouting to test its functional role in temporal lobe epileptogenesis. Targeting axonal growth cones may be an effective strategy to block mossy fiber sprouting. L-type calcium channels and calcineurin, a calcium-activated phosphatase, are critical for normal growth cone function. Previous studies have provided encouraging evidence that blocking L-type calcium channels or inhibiting calcineurin during epileptogenic treatments suppresses mossy fiber sprouting.
Rats were treated systemically with pilocarpine to induce status epilepticus, which lasted at least 2 hours. Then, osmotic pumps and cannulae were implanted to infuse calcineurin inhibitors (FK506 or cyclosporin A) or an L-type calcium channel blocker (nicardipine) into the dorsal dentate gyrus. After 28 days of continuous infusion, extent of mossy fiber sprouting was evaluated with Timm-staining and stereological methods.
Percentages of volumes of the granule cell layer + molecular layer that were Timm-positive were similar in infused and noninfused hippocampi.
These findings suggest inhibiting calcineurin or L-type calcium channels does not block mossy fiber sprouting in the pilocarpine-treated rat model of temporal lobe epilepsy.
Temporal lobe epilepsy is the most common form of epilepsy in adults (Engel et al., 1997), yet its pathogenesis remains unclear. Epileptogenic injuries result in sprouting of granule cell axons (mossy fibers) in the dentate gyrus of patients (Sutula et al., 1989; de Lanerolle et al., 1989; Houser et al., 1990; Babb et al., 1991) and rodent models of temporal lobe epilepsy (Nadler et al., 1980; Mello et al., 1993; Nissinen et al., 2000). However, the role of mossy fiber sprouting in generation of spontaneous seizures is controversial and unresolved. It has been proposed as a compensatory mechanism that restores excitatory synaptic input to inhibitory interneurons (Sloviter, 1992) or to create an aberrant positive-feedback circuit that reduces seizure threshold (Tauck and Nadler, 1985). To test these hypotheses it would be useful to selectively block mossy fiber sprouting. Prior attempts neutralized nerve growth factor with antibodies (Holtzman and Lowenstein, 1995), inhibited neural activity with tetrodotoxin (Buckmaster, 2004b), and blocked protein synthesis with cycloheximide around the time of epileptogenic injury (Williams et al., 2002; Toyoda and Buckmaster, 2005), but none prevented mossy fiber sprouting (cf. Longo and Mello, 1997; 1998).
An alternate approach is to directly target growth cone function. Presumably, mossy fiber sprouting begins with formation of growth cones on granule cell axons. Internal calcium concentration plays a critical role in normal growth cone function (reviewed in Henley and Poo, 2004; Gomez and Zheng, 2006; Wen and Zheng 2006). Neurite outgrowth is stimulated by calcium influx through voltage-gated calcium channels (Williams et al., 1992; Kuhn et al., 1998; Hong et al., 2000). L-type calcium channels are clustered on growth cones (Silver et al., 1990), and their blockade suppresses aberrant mossy fiber sprouting in hyperexcitable hippocampal slice cultures (Ikegaya, 1999). Repetitive, systemic administration of the L-type calcium channel blocker nicardipine was reported to suppress mossy fiber sprouting following pilocarpine-induced status epilepticus in mice (Ikegaya et al., 2000).
A rise in intracellular calcium concentration activates the protein phosphatase calcineurin (Klee et al., 1979). Calcineurin activity stimulates or inhibits neurite outgrowth, depending on cell type (Ferreira et al., 1993; Lyons et al., 1994; Chang et al., 1995; Lautermilch and Spitzer, 2000; Graef et al., 2003). The calcineurin-inhibitor FK506 was reported to inhibit kindling (Moia et al., 1994) and block mossy fiber sprouting in rats (Moriwaki et al., 1996). Thus, previous studies suggest inhibiting calcineurin or blocking L-type calcium channels may be useful to block mossy fiber sprouting. In the present study, we tested the ability of focal and continuous infusion of calcineurin inhibitors (FK506 or cyclosporin A) or an L-type calcium channel blocker (nicardipine) to suppress mossy fiber sprouting in rats after pilocarpine-induced status epilepticus.
All experiments were approved by the Stanford University Institutional Animal Care and Use Committee and performed in accordance with National Institutes of Health Guidance for the Care and Use of Laboratory Animals. Pilocarpine treatment was performed as described previously (Buckmaster, 2004a). Briefly, male Sprague-Dawley rats 34–52 days old were treated with atropine methylbromide (5 mg/kg i.p.), then 20 minutes later with pilocarpine hydrochloride (380 mg/kg i.p.) to induce status epilepticus. All chemicals were from Sigma-Aldrich (St. Louis, MO, U.S.A.) unless specified otherwise. After 2 hours of status epilepticus, seizures were suppressed with diazepam (10 mg/kg i.p., repeated as needed; Abbott Laboratories, North Chicago, IL, U.S.A.), and lactated ringers was administered subcutaneously. Control rats were treated identically, except they did not develop status epilepticus.
Osmotic pumps and cannulae were implanted to focally and continuously deliver drug to the dorsal, left dentate gyrus. Rats were anesthetized with 2% isoflurane (Baxter, Dearfield, IL, U.S.A.). Body temperature was monitored and controlled with a heating pad with feedback control. Rats were placed in a stereotaxic apparatus and their head and dorsal neck prepared for aseptic surgery. A scalp incision was made and < 1 mm diameter hole drilled through the skull 4.6 mm caudal and 2.8 mm left of bregma. A 3.5-mm-long 28-gauge cannula (Alzet brain infusion kit II; Durect Corporation, Cupertino, CA, U.S.A.) was inserted and secured to the skull with cranioplastic cement and a jeweler’s screw. An osmotic pump (model 2004; Durect Corporation) and tubing leading to the cannula were implanted subcutaneously over the back. Pumps contained 1 mM nicardipine, 1 mM FK506 (A.G. Scientific, San Diego, CA, U.S.A.), or 50 mM cyclosporin A (Alexis Biochemicals, San Diego, CA, U.S.A.) in vehicle solution containing 50% DMSO, 15% ethanol, and 0.1% fluorescein (Molecular Probes, Eugene, OR, U.S.A.), which was delivered at 0.25 µl/h for 28 days. Drug concentrations were at least 100X concentrations that effectively inhibit neurite outgrowth in vitro: 50,000X effective in vitro concentration of cyclosporin A (Ferreira et al., 1993; Chang et al., 1995; Lautermilch and Spitzer, 2000), 1000X effective in vitro concentration of FK506 (Chang et al., 1995), and 100–500X effective in vitro concentration of nicardipine (Ikegaya et al., 1997; 2000; Ikegaya, 1999). Infusions began 1–8 hours (means: 4.0, 5.8, and 5.3 hours for FK506, cyclosporin A, and nicardipine, respectively) after administering the first dose of diazepam, which was 2 hours after the onset of status epilepticus.
After 28 days of continuous infusion, rats were perfused and hippocampi sectioned and processed as described previously (Buckmaster, 2004b; Toyoda and Buckmaster, 2005). Briefly, rats were killed by urethane overdose (2 g/kg i.p.), perfused through the ascending aorta at 30 ml/min for 2 min with 0.9% sodium chloride, 5 min with 0.37% sodium sulfide, 1 min with 0.9% sodium chloride, and 30 min with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brain post-fixed overnight at 4°C. Then, hippocampi were isolated, cryoprotected in 30% sucrose in 0.1 M PB, gently straightened, frozen, and sectioned transversely with a microtome set at 30 µm. Starting at a random point near the septal pole, a 1-in-6 series of sections was mounted, dried, and developed for 45 min in 120 ml 50% gum arabic, 20 ml 2 M citrate buffer, 60 ml 0.5 M hydroquinone, and 1 ml 19% silver nitrate. Infused and noninfused hippocampi from the same animal developed together. Another 1-in-6 series was mounted, dried, coverslipped with Vectashield (Vector Laboratories, Burlingame, CA, U.S.A.), and checked for fluorescein-labeling to verify delivery of pump contents in infused hippocampi.
Mossy fiber sprouting was measured as described previously (Buckmaster and Dudek, 1997; Buckmaster, 2004b; Toyoda and Buckmaster, 2005). Briefly, sections were analyzed using a light microscope equipped with a 10X objective, Lucivid (MicroBrightField, Williston, VT, U.S.A.), and Neurolucida software (MicroBrightField). An investigator blind to the rats’ treatment made contours around the granule cell + molecular layer and Timm-positive parts of the granule cell + molecular layer of each section in the 1-in-6 series of each hippocampus. Drawing borders around Timm-stained areas can be subjective. Therefore, the same investigator evaluated all sections of all animals for a given drug treatment group. The Cavalieri method was used to estimate volumes of the granule cell layer + molecular layer and Timm-positive fraction. Extent of mossy fiber sprouting varies among individual rats treated systemically with chemoconvulsants (Buckmaster and Dudek, 1997). To avoid this potential confound the contralateral noninfused hippocampus served as a control in each rat.
To test whether focal infusion of FK506 had the desired inhibiting effect on calcineurin activity, Western blot analysis was used to measure inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which is expressed in endoplasmic reticulum of neurons and regulated by calcium entry through L-type channels and calcineurin activity (Genazzani et al., 1999; Graef et al. 1999). Five, naïve, male Sprague-Dawley rats (33 days old) were implanted for FK506 infusion as described above. After 14 days of infusion, rats were killed by urethane overdose (2 g/kg i.p.) and decapitated. Brains were quickly removed from the skull, placed in a chilled rat brain matrix (ASI Instruments, Warren, MI), and blocked coronally to isolate a 3 mm thick anterior-posterior segment that contained the infusion site. On a chilled platform, infused and contralateral hippocampi were isolated and immediately frozen and preserved at −70°C.
Sample preparation and Western analysis were conducted using techniques similar to those published previously (Genazzani et al., 1999). Tissue samples were homogenized in 320 mM sucrose in 5 mM Tris-HCl (pH 7.5) with 5 µg/ml pepstatin, antipain, and leupeptin. A crude membrane fraction was obtained after centrifuging the postnuclear supernatant at 12,000 × g for 10 min at 4°C. Protein concentrations were determined using a Micro BCA protein assay kit (Pierce, Rockford, IL), and aliquots were stored at −70°C until further use. Expression levels of IP3R1 protein were measured by Western blotting. Crude membrane proteins (6.25 µg) were loaded on 4–20% SDS-polyacrylamide gels and electrophoresed at 20 mA/gel for 110 min, before being transferred onto polyvinylidene difluoride (PVDF) membranes at 200 mA for 108 min. The blotted PVDF membrane was blocked with freshly prepared phosphate buffered saline (PBS) containing 5% nonfat milk and 0.025% sodium azide. The PVDF membrane was incubated in mouse anti-IP3R1 antibody (1:3, mouse monoclonal tissue culture supernatant, clone L24/18, UC Davis/NINDS/NIMH NeuroMab Facility) in above blocking solution overnight with agitation at 4°C. Following a wash, the PVDF membrane was incubated in peroxidase-labeled anti-mouse antibody (1:10,000; GE Healthcare, Piscataway, NJ) in PBS for 50 min at room temperature with agitation. The PVDF membrane was subsequently washed with 0.1% Tween 20 in PBS. ECL Western blotting detection reagents and autoradiography film (Hyperfilm ECL, GE Healthcare, Piscataway, NJ) were used for band detection. After completing analysis of IP3R1 bands, blotted PVDF membranes were washed in Restore Western blot stripping buffer (Pierce) for 15 minutes at room temperature and incubated with anti-actin, using protocols similar to those described above. IP3R1 and actin levels were quantified by densitometry using NIH image software. Ratiometric data for each animal, consisting of duplicate sample tubes, were averaged together.
Results from infused and noninfused hippocampi were compared with paired t tests. Results from different animals were compared with unpaired t tests. A value of p < 0.05 was considered significant. Values are reported as mean ± s.e.m.
Fifteen pilocarpine-treated rats experienced at least 2 hours of status epilepticus, and four did not. In all rats, fluorescein-labeling verified drug delivery into the left dentate gyrus (Figure 1). Fluorescence was very intense near cannulae tips and more diffuse and homogeneous extending throughout sections, including dentate gyrus, but not in contralateral hippocampal sections.
Western analysis was used to quantify levels of IP3R1 protein expressed in hippocampus around the site of FK506-infusion and contralaterally (Figure 2). Crude membrane was analyzed to assess the expression of the 300 kDa IP3R1 protein relative to actin. Average expression of IP3R1 relative to actin was 0.34 ± 0.03 in infused hippocampi, which was 50% of contralateral noninfused hippocampi (0.67 ± 0.08, p = 0.007).
Six rats that experienced status epilepticus and four that did not were infused with 1 mM FK506. All displayed black Timm staining in the hilus and stratum lucidum of CA3. All rats that experienced status epilepticus displayed aberrant mossy fiber sprouting into the granule cell and molecular layers of dentate gyrus (Figure 3). In contrast, pilocarpine-treated rats that did not experience status epilepticus did not display mossy fiber sprouting. For entire hippocampi (infused and noninfused), a larger percentage of the granule cell layer + molecular layer was Timm-positive in FK506-infused rats that experienced status epilepticus (9.4 ± 1.4%) than in those that did not (0.6 ± 0.2%; p = 0.001). In rats that had experienced status epilepticus, similar percentages of the granule cell layer + molecular layer were Timm-positive in infused (9.5 ± 1.3%) and noninfused hippocampi (9.4 ± 1.5%; p = 0.83). The section sampling interval was quite adequate for estimating volumes of Timm-positive granule cell layer + molecular layer, because mean coefficients of error of contour areas in individual hippocampi, calculated as described by West et al. (1991), were < 0.035. To determine if FK506 infusion had a focal effect that may have been missed by analyzing entire hippocampi, percentages of the granule cell layer + molecular layer that were Timm-positive were plotted with respect to distance of sections from infusion site. Average percent granule cell layer + molecular layer that was Timm-positive in sections ± 600 µm from infusion site (11.1 ± 1.1%) was similar to that of corresponding septotemporal regions in noninfused hippocampi (10.8 ± 1.2%; p = 0.24). These findings suggest FK506 did not block mossy fiber sprouting.
To test another inhibitor of calcineurin, 4 rats that experienced status epilepticus were infused with 50 mM cyclosporin A (Figure 4). For entire hippocampi, similar percentages of the granule cell layer + molecular layer were Timm-positive in infused (7.4 ± 1.5%) and noninfused hippocampi (8.1 ± 1.6%; p = 0.11). Mean coefficients of error were < 0.03. Average percent granule cell layer + molecular layer that was Timm-positive in sections ± 600 µm from infusion site (9.0 ± 0.7%) was similar to that of corresponding septotemporal regions of noninfused hippocampi (10.2 ± 0.9%; p = 0.27).
To determine whether blocking L-type calcium channels would prevent mossy fiber sprouting, 5 rats that experienced status epilepticus were infused with 1 mM nicardipine (Figure 5). Nicardipine did not significantly affect percent of granule cell layer + molecular layer that was Timm-positive in entire hippocampi (infused, 16.5 ± 2.7%; noninfused 15.5 ± 2.9%; p = 0.47; mean coefficients of error < 0.035). Average percent of granule cell layer + molecular layer that was Timm-positive in sections ± 600 µm from the infusion site (14.3 ± 2.7%) was similar to that of the corresponding septotemporal region of noninfused hippocampi (15.4 ± 2.9%; p = 0.15).
Focal and continuous infusion of FK506, cyclosporin A, or nicardipine did not significantly reduce percent of Timm-staining in the granule cell layer + molecular layer compared with contralateral noninfused hippocampi. These findings suggest inhibiting calcineurin or blocking L-type calcium channels does not suppress mossy fiber sprouting in the pilocarpine-treated rat model of temporal lobe epilepsy. These results are surprising in light of previous studies (Moriwaki et al., 1996; Ikegaya, 1999; Ikegaya et al., 2000) and disappointing, because they suggest targeting calcineurin or L-type calcium channels will not be helpful for evaluating functional effects of mossy fiber sprouting.
Why wasn’t mossy fiber sprouting blocked in the present study? One possibility is that insufficient drug reached sprouting mossy fibers. We cannot completely exclude this possibility, but it seems unlikely for the following reasons. First, the same osmotic pump and cannula system was used previously to effectively infuse drugs into the rat dentate gyrus (Galvan et al., 2000; Buckmaster, 2004b; Toyoda and Buckmaster, 2005). In the present study, fluorescein verified delivery of pump contents to the dentate gyrus. Presence of fluorescein does not guaranty continual infusion for 28 days, however. Second, pumps were loaded with high concentrations of drugs that were at least 100X concentrations that effectively inhibit neurite outgrowth in vitro. Using the same osmotic pump and cannula system, tetrodotoxin at only 10X its effective in vitro concentration blocked neuronal activity in dentate gyrus (Galvan et al., 2000). Third, previous studies that used osmotic pumps from the same manufacturer as those in the present study demonstrated delivery of biologically active FK506 and cyclosporin A for prolonged periods in vivo (Stepkowski et al., 1989; Shirbacheh et al., 1999). Finally, the same FK506 infusion dose and method significantly reduced IP3R1 expression. In cerebellar granule cells, IP3R1 gene and protein expression are regulated by calcium entry through L-type channels and inhibited by FK506 and cyclosporin A, implicating calcineurin as a regulator (Genazzani et al. 1999). Similarly, in hippocampal neurons FK506 and cyclosporin A inhibit expression of IP3R1 protein (Graef et al. 1999). Therefore, significantly lower ratios of IP3R1-to-actin in FK506-infused versus contralateral noninfused hippocampi suggest the approach used in the present study reduced calcineurin activity. Thus, it seems likely that active drugs were delivered to dentate gyrus at sufficient concentrations to test their potential effects on mossy fiber sprouting.
Another possible explanation for different conclusions of the present and previous studies is use of different experimental models. An amygdala-kindling model was used to evaluate effects of FK506 on mossy fiber sprouting (Moriwaki et al., 1996). In that study, rats were treated with FK506 during the kindling process. Therefore, it is unclear whether differences in mossy fiber sprouting are attributable to direct effects of FK506 or indirect effects on afterdischarge duration, neuron loss, etc. The present study avoided this confounding factor by delaying onset of drug infusion until after rats experienced at least 2 hours of status epilepticus. Hippocampal slice cultures chronically exposed to picrotoxin were reported to develop aberrant mossy fiber sprouting into the dentate gyrus molecular layer, which was suppressed by L-type calcium channel blockers (Ikegaya, 1999). However, mechanisms underlying mossy fiber sprouting in vitro might differ from those in more mature tissue in vivo. Repetitive, systemic administration of nicardipine was reported to suppress mossy fiber sprouting following pilocarpine-induced status epilepticus in mice (Ikegaya et al., 2000). However, animals in that study were treated with pilocarpine when they were only 14 days old, an age resistant to seizure-induced cell loss in rodents (Riviello et al., 2002; Raol et al., 2003). Extent of mossy fiber sprouting is correlated with degree of hilar neuron loss (Babb et al., 1991; Masukawa et al., 1996; Buckmaster and Dudek, 1997; Nissinen et al., 2001). Density of hilar neurons in pilocarpine-treated 14 day old mice was not significantly reduced (Ikegaya et al., 2000), and extent of mossy fiber sprouting in positive controls in that study appeared to be much less than that reported for adult mice treated with pilocarpine (Shibley and Smith, 2002). In the present study, more mature rats were treated with pilocarpine, and those that experienced status epilepticus developed robust mossy fiber sprouting, which provided a substantial baseline to evaluate potential blockade. Unfortunately, neither inhibiting calcineurin or blocking L-type calcium channels significantly reduced aberrant Timm staining. Nevertheless, this experimental paradigm can be used to test other candidate drugs.
Supported by NIH/NINDS and NIH/NCRR. The monoclonal antibody Anti-IP3 receptor type 1 was developed by and/or obtained from the UC Davis/NINDS/NIMH NeuroMab Facility, supported by NIH grant U24NS050606 and maintained by the Department of Pharmacology, School of Medicine, University of California, Davis, CA 95616. We confirm that we have read the Journal’s position on issue involved in ethical publication and affirm that this report is consistent with those guidelines.
None of the authors has any conflict of interest to disclose.