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0022-3565/97/2801-0471$03.00/0THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 280:471–476, 1997 Epileptic Activity Prevents Synapse Formation of HippocampalMossy Fibers via L-Type Calcium Channel Activation In Vitro YUJI IKEGAYA, MASATOSHI YOSHIDA, HIROSHI SAITO and NOBUYOSHI NISHIYAMA Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113, Japan Accepted for publication September 13, 1996 ABSTRACT
Hippocampal slice from early postnatal rat was used to eluci-
observations using a Timm method, a histochemical technique date the influence of epileptic activity elicited by picrotoxin on that preferentially labels synaptic terminals of mossy fibers, synapse formation of mossy fibers. Neurite reelongation and revealed that picrotoxin prevented synaptogenesis in the CA3 synaptogenesis of mossy fibers transected at 8 days in vitro region. This inhibitory effect of picrotoxin was completely abol- were confirmed by staining with DiI, a fluorescent membrane ished by tetrodotoxin or nicardipine (10 mM), a L-type calcium dye used as a neuronal tracer, and by recording field excitatory channel blocker, but not by 2-amino-5-phosphonopentanoic postsynaptic potentials (fEPSP) in the CA3 region evoked by acid (50 mM), a N-methyl-D-aspartate receptor antagonist, sug- stimulation of the dentate gyrus. Picrotoxin (50 mM), which gesting that influx of calcium ion via L-type calcium channels evoked spontaneous epileptiform firing in the CA3 region that during epileptic bursts mediated the disturbance of appropriate was occluded by tetrodotoxin (1 mM), hindered development of synapse formation of mossy fibers.
fEPSP amplitude after a lesion of mossy fibers. Furthermore, Because ontogenetic maturation of several regions in the that often demonstrate aberrant sprouting of mossy fibers central nervous system extends until early postnatal period, into the inner molecular layer of the dentate gyrus (Babb et certain forms of injury or disease during this critical stage al., 1991, Mathern et al., 1994) or massive reduction in the are correlated with developmental disorders. It is well known number of dendritic spines (Mu ¨ ller et al., 1993), it has not that epilepsy, frequency of which is much higher in children been reported whether epilepsy has any influences on neurite than in adults, particularly in the first year of life, is associ- outgrowth and synaptogenesis of mossy fibers during their ated with a broad spectrum of cognitive deficits when it developmental period. Fortunately, some recent reports occurs in this postnatal period (Alpherts and Aldenkamp, showed that developmental and physiological properties of 1990; Mizrahi, 1994; Stafstrom, 1995). However, few previ- mossy fibers were retained in organotypic slice cultures of ous reports identified characteristic changes in structure or postnatal hippocampus (Dailey et al., 1994; Robain et al., function of the central nervous system of epileptic patients, 1994; Frotscher et al., 1995). Therefore, we have asked which may underlie such cognitive deficits.
whether epileptic activity disturbs normal neurite outgrowth Hippocampal mossy fiber tract, axons projecting from the and synaptogenesis of mossy fibers using hippocampal slice granule cells in the dentate gyrus mainly to the pyramidal culture. As a result, we found severe suppression of synapse cells in the CA3 region, is formed very late because the formation of mossy fibers by epileptic activity.
dentate granule cells generate postnatally (Stirling andBliss, 1978; Amaral and Dent, 1981, Gaarskjær, 1986). This tract is believed to be involved in cognition and learningbecause its degeneration produces memory deficits (Conrad Preparation of organotypic slice cultures. For preparation of
and Roy, 1993; Vaher et al., 1994) and its synapses demon- hippocampal slices, postnatal 8 day (P8) Wistar rats were decapi-tated and the brains were removed. The hippocampi were cut into strate a high degree of functional plasticity (Bradler and 300-mm thick slices in cold glucose-enriched Gey's buffer and were Barrionuevo, 1989; Mitsuno et al., 1994; Malenka, 1995).
then cultivated according to the method introduced by Stoppini et al. Although there are numerous reports concerning dynamic (1991). Briefly, selected sections were placed on moistened translu- morphological plasticity of mossy fibers in epileptic seizure cent membranes (0.4 mm Culture Plate Insert, 30 mm diameter,Millicell-CM, Millipore Corporation, Bedford, MA) that were in-serted in six-well plates (35 mm in diameter) filled with 1 ml of Received for publication April 18, 1996.
medium (50% minimum essential medium, 25% Hanks' balanced ABBREVIATIONS: ACSF, artificial cerebrospinal fluid; AP5, 2-amino-5-phosphonopentanoic acid; DIV, day in vitro; fEPSP, field excitatory
postsynaptic potential; GABA, g-aminobutyric acid; NMDA, N-methyl-D-aspartate; DiI, 3,39-dilinolenyloxacarbocyanin perchlorate.

Ikegaya et al.
salt solution, 25% heat inactivated horse serum). The cultures were same ACSF. The hilus of the upper blade of the dentate granule cell kept at 36°C in a humidified, CO -enriched atmosphere. The culture layer was stimulated with a bipolar electrode. The evoked potential medium was changed twice a week.
was extracellularly recorded from the CA3 pyramidal cell layer with Lesioning of mossy fiber tract. In some slices, mossy fibers
a glass capillary microelectrode filled with 0.9% NaCl. Positive field were transected at 8 DIV along the line linking the lips of the upper potential (see fig. 1, B, F and H) reflected fEPSP because it was and lower blade of the granule cell layers (see fig. 1, C, E and G, fig.
blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (10 mM), a non- 5, B, C and D). The lesion was performed under an operating micro- NMDA receptor antagonist (data not shown). The maximal size of scope using a manipulator with a razor blade.
fEPSP was used as an index of the number of functional synaptic DiI labeling. Cultured slices were fixed with 0.1 M phosphate
contacts formed as a function of time after a lesion (Muller et al., buffer containing 4% paraformaldehyde 1 day after DiI crystal was 1993; Stoppini et al., 1993).
placed on the dentate gyrus. After a 5-wk incubation in the fixative Timm staining. For Timm stain, cultures were washed with 0.1
at room temperature, the DiI-labeled axons were observed using a M phosphate buffer and were then immersed for 10 min in 0.37% fluorescent microscope (Honig and Hume, 1989).
sodium sulfide solution, immediately followed by fixation for 15 min Extracellular recordings. Cultured slices were submerged for
with 10% (v/v) formaldehyde solution. After washed with 0.1 M 30 to 60 min in ACSF, which was composed of 124 mM NaCl, 5.0 mM phosphate buffer, the cultures were dehydrated with 70 and 96% KCl, 2.4 mM CaCl , 1.3 mM MgSO , 1.24 mM KH PO , 26.0 mM ethanol, and dried. To perform the sulfide silver staining, they were NaHCO and 10.0 mM glucose and was saturated with 95% O -5% submerged in the physical developer according to the method of CO , and were transferred into a recording chamber filled with the Sloviter (1982) and were then incubated in a dark room for 50 min at 26°C. The slices were washed with distilled water at the end of thereaction.
Drugs. In slice cultures, the drugs were applied in the culture
medium on and after 8 DIV. For recording spontaneous activities,the drugs were dissolved in ACSF. All the drugs used were obtained from commercial sources; picrotoxin (Wako Pure Chemical Industry,Ltd., Osaka, Japan), a GABA receptor channel blocker; tetrodotoxin(Sigma Chemical Co., St. Louis, MO), a voltage-sensitive sodiumchannel blocker; nicardipine (Wako), a L-type calcium channel block-er; AP5 (Sigma), a NMDA receptor antagonist; 6-cyano-7-nitroqui-noxaline-2,3-dione (Research Biochemical Incorporated, Natick,MA), a non-NMDA receptor antagonist.
Mossy fiber growth and synapse formation after a
lesion. In a series of these experiments, we investigated the
effect of epileptic activity on reformation of synapses after a
section of maturated mossy fibers because it was difficult to
know exactly when mossy fiber formation starts in vivo.
Many previous reports adopting this tissue lesion method
indicated that organotypic characteristics, developmental
processes and neuronal properties in vivo are well-preserved
in hippocampal slice cultivated after the lesion (Ga¨hwiler
and Brown, 1985; Zimmer and Ga¨hwiler, 1987, Heimrich and
Frotscher, 1993; Li et al., 1993; Stoppini, et al., 1993; Dailey
et al., 1994; Frotscher et al., 1995).
Mossy fibers were lesioned at 8 DIV because cultured slices were electrophysiologically stabilized by this time (fig. 2,open circle). First, we examined reelongation and synapto- Fig. 1. Lesion-induced reorganization of mossy fibers. Fluorescent im-
ages of hippocampal slices stained with DiI were observed at 8 DIV (A),
0 day (C) or 7 days (E) after lesions at 8 DIV. White lines demonstrate the
granule cell layer of the dentate gyrus (DG) and the pyramidal cell layer
of the CA1-4 regions. Mossy fibers were transected along white broken
lines. A scale bar represents 1 mm. Right traces were typical field
potentials (average of four) in the CA3 region at 8 DIV (B), 0 day (D) or
7 days (F) after the lesion. The dentate gyrus was stimulated at the time
Fig. 2. Changes in amplitude of fEPSP recorded in the CA3 region
indicated by arrows. DiI image in G and field potential in H were elicited by supramaximal stimulation of the dentate gyrus in intact obtained from slices treated with picrotoxin (50 mM) for 7 days after slices (n 5 5–9) (open circle) or slices lesioned at 8 DIV (n 5 6 –9) (closed lesions at 8 DIV.
circle). Each point represents mean 6 S.E.M.
Epilepsy and Hippocampal Maturation
genesis of transected mossy fibers under our culture condi- Effect of picrotoxin on mossy fiber synapse forma-
tions. Neurite outgrowth was observed by staining with DiI, tion. For evaluating the influence of epileptic activity on
which is a fluorescent membrane dye used as a neuronal synapse formation of mossy fibers, picrotoxin was added to tracer (fig. 1, A, C, E and G). Although mossy fibers were culture medium at a concentration of 50 mM immediately completely transected by the method adopted in this study after the lesion. Development of fEPSP amplitude after the (fig. 1C), they elongated close to the pyramidal cell layer of section of mossy fibers was prevented in slices cultivated in the CA3 region beyond the transection at 7 days after the medium containing picrotoxin (figs. 1H and 4A). This inhib- section (fig. 1E), and formed functional excitatory synapses itory effect of picrotoxin was completely abolished by appli- on the pyramidal cells, which was estimated by recording cation of tetrodotoxin (1 mM) (fig. 4B). Picrotoxin did not synaptic responses reflecting fEPSP in the CA3 region reduce fEPSP amplitude in intact slices (maximal response evoked by stimulation of the dentate gyrus (fig. 1, B, D and amplitudes in nontransected slices cultivated for 7 days in F). Because fEPSP in the CA3 region was not observed im- medium containing picrotoxin was 2.52 6 0.29 mV, and that mediately after the lesion (n 5 32) (fig. 1D), it was again in normal medium was 2.13 6 0.42 mV; means 6 S.E.M. of confirmed that all mossy fibers were transected. At more seven or six slices, respectively). To determine whether spon- than 4 days after the lesion, however, fEPSP appeared in all taneous activity in normal medium, which was rarely seen as 82 slices tested. A change in the maximal size of evoked above described, contributed to the recovery of fEPSP after synaptic responses was shown in figure 2. For promoting the lesion, slices were cultivated in the medium containing comparisons, a change in fEPSP amplitude in nontransected tetrodotoxin for 7 days after the lesion. Tetrodotoxin (1 mM) slices was superimposed on the same figure. An extent of did not affect fEPSP amplitude (maximal response ampli- maximal response in slices at 14 days after the section was tudes in slices cultivated in normal medium for 7 days was similar to that in DIV-matched intact slices.
2.37 6 0.57 mV, and that in medium containing tetrodotoxin Epileptic activity. Although epileptiform burst discharge
was 1.94 6 0.58 mV; means 6 S.E.M. of eight or nine slices, can be elicited in acutely prepared hippocampus slices and respectively). Inhibition of synaptogenesis by continuous ep- cultured slices in a number of diverse ways, a simple proce- ileptic activities was also confirmed with a Timm method, a dure is to block inhibitory postsynaptic potentials mediatedby GABA with its receptor antagonist (Dichter and Ayala,1987; Thompson and Ga¨hwiler, 1992). At 8 DIV, 42 of 43slices (97.7%) exposed to picrotoxin (50 mM), a GABA recep- tor channel blocker, showed spontaneous synchronized epi-leptiform bursts with a high regularity (2.05 6 0.47 bursts/min; mean 6 S.E.M. of eight slices) in the CA3 region, whichindividually consisted of 7.87 6 0.85 (mean 6 S.E.M. of eightslices) repetitive firings (fig. 3B), although epileptiform ac-tivity was not observed in normal ACSF (fig. 3A). Only 2 in 82intact slices tested (2.4%) exhibited spontaneous activity thatconsisted of a single, but not repetitive, firing. The epilepticbursts induced by picrotoxin was blocked by application oftetrodotoxin (1 mM) (fig. 3C).
Fig. 4. Inhibitory effect of picrotoxin on mossy fiber synapse forma-
tion. A, Change in size of maximal fEPSP were observed in hippocam-
pal slices cultivated in normal medium (n 5 5–11) (open circle) or in
medium containing picrotoxin (PTX, 50 mM) (n 5 5–9) (closed circle). B,
Field potentials were recorded at 7 days after the lesion in slices
cultivated in normal medium (n 5 12) (open column), in picrotoxin (50
mM, n 5 6) (hatched column) or in coexistence of picrotoxin withtetrodotoxin (TTX, 1 mM, n 5 6), nicardipine (Nic, 10 mM, n 5 7) or Fig. 3. Typical records of epileptic activity in the CA3 region of a
2-amino-5-phosphonopentanoic acid (AP5, 50 mM, n 5 7) (closed hippocampal slice at 8 DIV. Field potentials were recorded in normal column). Data are means 6 S.E.M. of 6 to 12 cases. *P , .05, **P , .01 ACSF (A), in ACSF containing picrotoxin (PTX, 50 mM) (B), containing vs. control, ##P , .01 vs. PTX: Tukey's test after analysis of variance picrotoxin (50 mM) and tetrodotoxin (TTX, 1 mM) (C), or containing (ANOVA). Data in A and B were obtained from different series of picrotoxin (50 mM) and nicardipine (Nic, 10 mM) (D). A burst indicated by experiments and were not pooled because deviation among experi- an arrow in Ba was expanded in Bb.
ments was large.

Ikegaya et al.
recovery of fEPSP from the lesion was blocked by nicardipine(10 mM) but not by AP5 (50 mM) (fig. 4B). The ameliorativeeffect of nicardipine against picrotoxin was also confirmedmorphologically by the Timm method (fig. 5D). We thenexamined if nicardipine altered the epileptiform activity in-duced by picrotoxin. Picrotoxin (50 mM) elicited the burstingeven in nicardipine- (10 mM) containing ACSF in all eightpatients tested (fig. 3D). This epileptiform activity showedhigh regularity and its frequency was 2.35 6 0.59 min21(means 6 S.E.M. of eight slices). Each burst was consisted of6.82 6 1.02 (means 6 S.E.M. of eight slices) repetitive fir-ings. These properties were very similar to those of burstsinduced in normal ACSF. We concluded, therefore, that ni-cardipine did not change the character of picrotoxin-elicitedbursts, consistent with a previous work reporting that dihy- dropyridine-type calcium channel blocker did not inhibit ep-ileptic discharge (van Luijtelaar et al., 1994). In addition,exposure of intact slices to nicardipine or AP5 in the absenceof picrotoxin from 8 DIV to 15 DIV did not affect fEPSPamplitude evoked in the CA3 region (data not shown, n 56–9). Taken together, it is suggested that calcium influx through L-type calcium channels during epileptic bursts me-diated the disturbance of appropriate synapse formation ofmossy fibers.
Fig. 5. Bright-field images of hippocampal slices stained with a Timm
method were obtained from an intact slice at 15 DIV (A) or slices
cultivated in normal medium (B), in picrotoxin (PIC, 50 mM) (C) or in
picrotoxin (50 mM) and nicardipine (Nic, 10 mM) (D) for 7 days after Using hippocampal slice culture, we demonstrated that lesions at 8 DIV. Mossy fibers were transected along white broken lines.
The area indicated by an arrow is stratum lucidum, where terminals of picrotoxin prevented reorganization of mossy fibers via L- mossy fiber tract form synapses on the CA3 pyramidal cells.
type calcium channel activation.
¨ ller et al. (1993) found that the amplitude of evoked histochemical technique that labels synaptic terminals of fEPSP was depressed after chronic application of GABAA mossy fibers because of their high zinc content (fig. 5). In receptor blockers. In our study, however, picrotoxin had no extrahippocampal area, subiculum and entorhinal cortex effect on fEPSP in intact slices. This apparent contradiction were also stained, consistent with a previous report showing may come from the following: 1) Cultures prepared with the that synapse boutons in these regions contained zinc (Slo- roller-tube method they used formed a monolayer explant mianka, 1992). In all 16 slices cultivated in normal medium, and might be more delicate than slices cultivated with the the stratum lucidum of the pyramidal cell layer in the CA3 static culture method we applied, which retained a few cell region, which is indicated by an arrow in figure 5A, was layers of thickness (Stoppini et al., 1991). The difference in stained across the transection (fig. 5B), but this was not slice cultivation procedures may also account for the discrep- observed in slices cultivated in picrotoxin in all 12 cases ancy in the extent of reinnervation in control cultures after examined (fig. 5C). DiI labeling technique revealed that pic- the lesion. Indeed, both our cultures and those of Stoppini et rotoxin-treated mossy fibers grew past the lesion into the al. (1993) showed 100% reinnervation, although in the pre- CA3 pyramidal cell layer at 7 days after the lesion (fig. 1G) in vious studies by Zimmer and Ga¨hwiler (1987) and Dailey et all nine slices tested. These results suggest that picrotoxin al. (1994) they could not produce such a high reinnervation did not block outgrowth but inhibited synaptogenesis of rate in slices obtained by roller-tube method. 2) Concentra- mossy fibers. Another consistent feature in hippocampal tion of picrotoxin Mu ¨ ller et al. (1993) applied was 500 mM slices treated with picrotoxin was aberrant sprouting of that was 10 times higher than ours and might exert nonspe- mossy fibers into the molecular layer of the dentate gyrus. In cific or toxic effects.
3 of 15 slices cultivated in control medium after the lesions, Barbin et al. (1993) reported that blockade of GABA this phenomenon was faintly observed (fig. 5B). This may be ceptors reduced neurite length of cultured hippocampal neu- due to temporary loss of target produced by lesions because rons and suggested the involvement of GABA receptors in some reports showed that loss of hilus interneurons, one of neurite outgrowth. Our result that picrotoxin inhibited syn- the main postsynaptic targets of mossy fiber tract, caused apse formation of mossy fibers can also be interpreted as a such aberrant sprouting (Babb et al., 1991).
consequence of prevented reelongation of transected mossy Epileptic bursts elicit sustained depolarization shift of neu- fibers. However, this possibility is ruled out by an observa- ronal membrane potential that may allow influx of calcium tion using DiI labeling technique that indicates that picro- ion via voltage-sensitive calcium channels or NMDA receptor toxin-treated mossy fibers extended close to the pyramidal channels. Finally, we tested the effects of nicardipine, a L- cell layer of the CA3 region at 7 days after a lesion. Although type calcium channel blocker, and AP5, a NMDA receptor we did not examine whether chronic application of picrotoxin antagonist, on picrotoxin-induced inhibition of synaptogen- produced epileptic activity in cultured slices, the inhibitory esis of mossy fibers. The inhibitory effect of picrotoxin on the effect of picrotoxin on synapse formation was probably due to Epilepsy and Hippocampal Maturation
epileptic activity per se because it was completely canceled by there are indications that reactive synaptogenesis may be tetrodotoxin. In addition, aberrant sprouting of mossy fibers involved in learning and memory (Greenough and Bailey, into the molecular layer of the dentate gyrus, that has been 1988; Moser et al., 1994). Therefore, our results may account typically observed in epileptic hippocampus (Babb et al., in part, for cognitive deficits elicited by childhood epilepsy, 1991, Mathern et al., 1994), was confirmed in picrotoxin- and further investigation using our method will provide fur- treated slices by a Timm method. This also suggests that ther insights and understandings with respect to this syn- picrotoxin actually elicited epileptiform activity in cultured slices. Taken together, these data strongly suggest that epi- Our results indicate that calcium ion influx through L-type leptic activity hindered lesion-induced reorganization of calcium channels may mediate a disorder of synapse forma- mossy fibers.
tion of mossy fibers, consistent with previous reports showing Represa et al. (1989) found that high affinity binding sites that calcium ion influx plays a major role in neuronal injury for kainate increased in the CA3 region of childhood epilep- associated with epilepsy (Wasterlain et al., 1993). However, tics. Although their result seems to contradict our finding, it many reports examining correlation between synaptogenesis is known that the type of neuronal firings often determine and calcium ion movement implied contradictive informa- the direction of plasticity. For example, the direction of the tion. Although most of these data demonstrate an essential synaptic gain change depends on the membrane discharge of role of calcium ion in synaptogenesis (Basarsky et al., 1994), the postsynaptic cell in the hippocampus (Artola and Singer, others suggest that synapse formation and increase in intra- 1993; Malenka, 1995). Thus, further detail examination on cellular calcium ion is irrelevant (Verderio et al., 1994). Our picrotoxin-induced bursts in cultured slice might elucidate observation that blockade of calcium ion influx abolished the difference between the preceding report and our finding.
epileptic activity-induced inhibition of synapse formation Recovery time course of maximal fEPSP amplitude after suggests a repressive role of calcium ion, which further com- mossy-fiber lesions approximately matched to that of intrin- plicated the discussion. One possible explanation is that ex- sic formation of mossy fibers, which are completed during cessive calcium concentration results in obstruction of syn- postnatal 1 to 2 wk (Stirling and Bliss, 1978; Amaral and aptogenesis although intermediate degree of calcium ion Dent, 1981; Gaarskjær, 1986). Moreover, the maximal fEPSP level may be required for it. Despite ambiguous role of cal- amplitude recorded at 14 days after the section recovered to cium ion in mossy fiber synaptogenesis, our results suggest a an extent comparable to that in DIV-matched intact slices.
novel protective action of L-type calcium channel blockers Additionally, synaptic terminals of regenerated mossy fibers against disturbance of normal synaptic maturation associ- were Timm-stain positive, that was one of the important ated with epileptic seizure, besides its antiepileptic proper- characteristics of mossy fibers. These observations strongly ties as have been proposed in various models of epilepsy (van support the idea proposed by several previous reports that Luijtelaar et al., 1994;, Straub et al., 1994). Further investi- developmental manner and organotypic nature in vivo are gations on this finding may endow valuable information for conserved in structures regenerated after the lesion (Ga¨h- applying calcium channel blockers as prophylactics against wiler and Brown, 1985; Heimrich and Frotscher, 1993; Li et cognitive deficits induced by childhood epilepsy.
al., 1993; Stoppini, et al., 1993; Frotscher et al., 1995). Ac- Finally, organotypic slice culture used in our study pre- cordingly, process and characteristics of reorganizing mossy serves in vivo nature to a high degree and renders a useful fibers after a lesion in our study may correspond to those of model for studying developmental cellular dynamics in a developmentally programmed formation of mossy fibers mammalian central nervous system.
(Zimmer and Ga¨hwiler, 1987; Dailey et al., 1994).
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