Brain Research 910 (2001) 199–203www.elsevier.com/ locate /bres

Short communication21Enhanced Ca influx with mossy fiber stimulation in hippocampal

CA3 neurons of spontaneously epileptic ratsa a a bTaku Amano , Hiroko Amano , Hiroaki Matsubayashi , Kumatoshi Ishihara ,

c a ,*Tadao Serikawa , Masashi SasaaDepartment of Pharmacology, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan

bDepartment of Pharmacotherapy, Institute of Pharmaceutical Science, Faculty of Medicine, Hiroshima University, Hiroshima 734-8551, JapancInstitute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

Accepted 29 May 2001

Abstract

21 21This study was performed to determine whether the intracellular Ca concentration ([Ca ] ) is increased in hippocampal CA3i

neurons of spontaneously epileptic rats (SER) which show both absence-like and convulsive seizures using hippocampal slices loaded21with Calcium Green-1 when a weak single stimulation is given to the mossy fiber. [Ca ] in the CA3 area was significantly increasedi

after a single stimulus to mossy fibers in SER, while no changes were detected in normal Wistar rats. These findings suggest the existence21of an abnormality in the Ca channel in the SER CA3 region and that this is probably responsible for epileptic seizures. 2001

Elsevier Science B.V. All rights reserved.

Theme: Excitable membranes and synaptic transmission

Topic: Calcium channel structure, function, and expression

Keywords: Calcium influx; Spontaneously epileptic rat (SER); Calcium Green-1; Hippocampal CA3 area; Mossy fiber

21The spontaneously epileptic rat (SER: tm/ tm, zi /zi), SER are suggested to be due to enhanced Ca influxes inobtained by mating a heterozygote tremor rat (tm/1) with the neurons. Thus, the present study was performed to

21a homozygote zitter rat (zi /zi), is a double mutant that determine whether the Ca influx is actually enhanced inshows both tonic convulsions and absence-like seizures the CA3 area of SER when a single stimulus is applied tocharacterized by the sudden appearance of 5–7 Hz spike- mossy fibers using hippocampal slices loaded with Cal-wave-like complexes in the cortical and hippocampal EEG cium Green-1.after 8–9 weeks of age [12,14,18]. Our previous electro- In our previous study [1], Fura-2 was used as aphysiological studies have demonstrated that SER has an fluorescent dye, as its ratiometric F /F excitation ratio340 380

21abnormal excitability of hippocampal CA3 neurons, which allows accurate measurement of the intracellular Cashows a long-lasting depolarization shift induced by a concentration, which is essentially independent of thesingle stimulation of mossy fibers, probably resulting from amount of dye within the cell, and thus immune to

21Ca channel abnormalities [7,10]. In addition, an increase problems such as uneven dye loading, leakage of dye and21 1of [Ca ] with high K solution application is more photobleaching, cellular autofluorescence, scattering back-i

pronounced in the hippocampal CA3 area of SER than that grounds and unequal slice thickness [5]. However, in this21of normal Wistar rats [1]. Ca channel abnormalities have study, we employed a sampling photograph at high speed

also been reported in hippocampal neurons of kindled (one frame per 5 ms) to examine the rapid changes in21animals [4,16]. Therefore, epileptic seizures observed in [Ca ] induced by single mossy fiber stimulation, a modeli

similar to that used for the previous electrophysiologicalstudy [7,10]. Therefore, we used a nonratiometric (single*Corresponding author. Tel.: 181-82-257-5140; fax: 181-82-257-wavelength) indicator, Calcium Green-1, which is a5144.

E-mail address: sasa@mcai.med.hiroshima-u.ac.jp (M. Sasa). fluorescent, long-wavelength (excitation 506 nm, emission

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )02700-7

200 T. Amano et al. / Brain Research 910 (2001) 199 –203

534 nm), fluo 3-like, calcium-sensitive indicator, to detect were obtained on the three areas of hippocampal CA3 (a,meager calcium transients at low indicator concentrations b, and c) pyramidal cell layers where three pixels were[15]. The maximum (F ) to minimum (F ) value of designated as square regions (1423142 mm) (Fig. 1A andmax min

21fluoresence (F /F ) is approximately 14 [17]. The D). The increases in [Ca ] as ratio values (D ratio), themax min i21 21Ca -binding affinity of Calcium Green-1, which is a latencies from the stimuli to the beginning of [Ca ]i

21high-affinity Ca indicator, is comparable to that of Fura- increases, and the peak latencies from the stimuli to the212: K values are 190 and 224 nm, respectively [17]. Such peak of [Ca ] were measured at a, b, and c of thed i

21indicators are known to buffer small changes in [Ca ] hippocampal CA3 area in SER and normal Wistar rats.i

when loaded into the cell at high concentrations [6]. Each value is indicated as the mean6S.E.M. TheHowever, when Calcium Green-1 at a concentration of 5 significance of the differences between SER and normal

21mM is added to a cell suspension, the changes in [Ca ] rats was determined by ANOVA analysis.i

are well detected [15]. In addition, it has been demon- When a single stimulus of 4 and 7 V was given to thestrated that there is a linear relationship between the mossy fibers of a SER and a normal Wistar Rat, a long-

21fluorescence measurement and free Ca [11]. Thus, we lasting depolarization shift accompanying repetitive firingused Calcium Green-1 for the measurement of small and a single action potential were observed, respectively,

21changes in [Ca ] following a single stimulation of the as demonstrated previously [7,10]. With a single stimulusi21mossy fibers. of 4 V applied to the mossy fiber, an increase in [Ca ]i

Ten hippocampal slices (400 mm thick) were prepared was observed in all three hippocampal CA3 regionsfrom five SER (three males and two females) aged 11–12 examined in SER, and then a gradual decrease to theweeks old when they exhibited both absence-like seizures original level within 1–2 s (Figs. 1C and 2A). Non-and tonic convulsion, and 10 slices from five age-matched ratiometric indicators such as Calcium Green-1 are knownWistar rats (three males and two females), according to the to be sensitive to photobleaching. However, we confirmedmethod of Kudo et al. [9] and Silei et al. [15]. Each slice that the same results could be obtained from a series of

21was loaded with 10 mM Calcium Green 1-AM (Molecular four successive measurements of the [Ca ] increase ini

Probes, USA), a 1:10 volume of detergent added (Cre- one slice using Calcium Green-1 at a concentration of 10mophore EL, Sigma, USA) for 90 min (258C), rinsed in mM, indicating that bleaching is negligible under ourartificial cerebrospinal fluid (ACSF) for a further 30 min, experimental conditions. The D ratio with stimulation ofand then placed in a recording chamber (approximately 1 areas a, b, and c of hippocampal CA3 in SER were

3cm ). The chamber was continuously perfused with ACSF 0.02060.012, 0.02760.015, and 0.04960.017, respective-at a rate of 2 ml /min at 258C. The ACSF was composed of ly (n 5 10, Table 1).

21(in mM): NaCl, 113; KCl, 3; CaCl , 2; MgCl , 1; The peaks of [Ca ] in areas b and c were significantly2 2 i

NaH PO , 1; NaHCO , 25; and glucose, 11; gassed with (P , 0.001) greater than those in area a. Moreover, the2 4 3

O 95%–CO 5%. A stimulus of 4 and 7 V with 0.1-ms latencies from the application of the stimulus to the2 221duration was applied to the mossy fiber through a bipolar beginning of the increase in [Ca ] in areas a, b and ci

stimulating electrode placed in the granule cell layer in the were all 19.767.3 ms (n 5 10, Table 1). These values diddentate gyrus of SER and normal Wistar rats, respectively not differ and were similar in the three areas examined.(Fig. 1A and D). The peak latencies from the stimuli in areas a, b and c

The experiments were started after sufficient perfusion were 39.9623.3, 42.8621.1, and 39.2612.6 ms (n 5 10,for stable conditions (at least 1 min). Changes in the Table 1), respectively. There were no significant differ-intensity of the fluorescence of Calcium Green-1 excited at ences among the three values. In contrast, increases in

21a wavelength of 480 nm (F ) were monitored every 5 ms [Ca ] were not observed in areas a, b and c in any of the480 i

for 5 s using a microscope with a four-fold fluor lens 10 slices from normal Wistar rats (Figs. 1F and 2B) even(Fluor 43 /0.13, Nikon, Japan), and equipped with a CCD when the stimulus to the mossy fiber was increased to 7 V.camera (C-6790-81, Hamamatsu Photonics, Japan). The In Fig. 2B, the traces for the normal rat do not decaystart of the recording position (time 0 ms), commenced within the experiment.after sufficient perfusion with ACSF, was taken as the In our previous electrophysiological studies, when aresting fluorescence intensity (F ). After stimulation of single stimulus was given to the mossy fibers, a long-480r

mossy fibers, each fluorescence intensity was recorded as lasting depolarization with repetitive firing, probably due21the stimulated fluorescence intensity (F ) every 0.5 ms. to Ca influx, was observed in the hippocampal CA3 of480s

To reduce the effects of the dye contents of the cell and SER with epileptic seizures, but not in that of normaluneven dye loading, each F value was then normalized Wistar rats. In the present study, an actual increase in480s

21to F every 5 ms for 5 s using an Aqua Cosmos/RATIO [Ca ] by a single stimulation given to mossy fibers was480r i

Imaging System attached to a digital fluorescence analyzer detected in hippocampal CA3 of SER. The fluorescence(Hamamatsu Photonics), and the ratio obtained was F / lifetime, which is the characteristic interval that a fluores-480s

F . The appropriate ratio ranges were designated auto- cent molecule remains in an excited state before recovering480r

matically with this system. The optical traces in the figures to the ground state, is relatively long: 1.05 and 3.06 h in

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Fig. 1. Traces of measured pixels on areas a, b, and c in the hippocampal CA3 regions, and the stimulating electrode in the dentate gyrus (S) of a SER (A) and a normal Wistar rat (D). Ratio imagesjust before a single stimulation in a SER (B) and a normal Wistar rat (E), and 40 ms after 4 V stimulation in a SER (C) and a normal Wistar rat (F).

202 T. Amano et al. / Brain Research 910 (2001) 199 –203

21Fig. 2. Representative time courses of the ratio of [Ca ] for a SER (A) and a normal Wistar rat (B). All tracings show typical examples obtained fromi

one slice of each animal.

21 21 21Ca -free and Ca -bound conditions, respectively [17]. animals. The increase in [Ca ] in normal animals isi

Therefore, the slow delay of the fluorescent signals seen in considered to be too small (or negligible) to be measured:21 21SER is considered to reflect intracellular Ca changes. in other words, the Ca channel may not have opened21 21The ability of Calcium Green-1 to release Ca from the wide enough for Ca to enter upon a single stimulus to

21intracellular Ca store remains unclear, as it does for the mossy fibers. The results are in accordance with our21Fura-2. However, Ca influxed from the outside could previous finding that single mossy fiber stimulation in-

21release Ca from the store. Therefore, the increase in duces a long-lasting depolarization shift in the hippocam-21[Ca ] after stimulation of the mossy fiber may be the pal CA3 neurons of SER, but not that of normal animalsi

21 21summation of influxed and released Ca . In contrast, [7,10]. Abnormally large amounts of [Ca ] were foundi21there were no increases in [Ca ] upon mossy fiber to be localized in the hippocampal CA3 areas of SER,i

stimulation in the hippocampal CA3 area of normal suggesting an epileptic focus may exist in the hippocampalCA3 area of SER.

21Table 1 Since such an increase in [Ca ] was not observed ini21Latency, D ratio and peak latency of the increases in [Ca ] ini the CA3 of normal Wistar rats even with a much higherhippocampal CA3 fields of SER and normal Wistar rats following a 21stimulus to the mossy fibers, the Ca channels of CA3single stimulus applied to mossy fibers

neurons in SER are considered to be easily opened by aAreas measured in CA3 region single stimulus to the mossy fibers. Glutamate releaseda b c from the mossy fiber terminals with a single stimulation is

1suggested to induce an action potential, carried by Na ,SER (n 5 10)21

Latency (ms) 19.767.3 19.767.3 19.767.3 resulting in easy opening of the voltage-dependent CaD ratio 0.02060.012* 0.02760.015 0.04960.017 channel of the CA3 neurons in SER.Peak latency (ms) 39.9623.3 42.8621.1 39.2612.6 In behavioral studies, a new dihydrothienopyridine

21Wistar rats (n 5 10) derivative Ca channel antagonist, methyl-4,7-dihydro-3-Latency (ms) – – – isobutyl-6-methyl-4-(3-nitrophenyl)-thieno-[2,3-D ratio 0 0 0 b]pyridine-5-carboxylate (S-312-d), which can easily reachPeak latency (ms) – – –

the brain, was actually effective in inhibiting seizures in21Latency (ms), time from stimulation to the beginning of the increase in SER [2]. Therefore, an abnormality of Ca channels, at21 21[Ca ] ; D ratio, the peak of [Ca ] from the baseline; peak latency (ms),i i

21 least in CA3 neurons of SER, is suggested to be respon-time from the stimulation to the peak of [Ca ] (see Fig. 2A). Each valueisible for the epileptic seizures in SER as observed inindicates the mean6S.E.M.

*P , 0.001, compared with the values of areas b and c (n 5 10). kindled animals, the tottering mouse [3,7,8,13,16].

T. Amano et al. / Brain Research 910 (2001) 199 –203 203

pocampal CA3 pyramidal neurons of spontaneously epileptic ratsAcknowledgements(SER), a double mutant, Exp. Neurol. 119 (1993) 287–290.

[8] G. Kostopoulos, C. Psarropoulou, Increased postsynaptic excitabilityThis study was supported, in part, by a Grant-in-Aid for in hippocampal slices from the tottering epileptic mutant mouse,

Scientific Research from the Ministry of Education, Sci- Epilepsy Res. 6 (1990) 49–55.ence, Sports and Culture, and a grant (No. 12470018) from [9] Y. Kudo, K. Ito, H. Miyakawa, Y. Izumi, A. Ogura, H. Kato,

Cytoplasmic calcium elevation in hippocampal granule cell inducedthe National Center of Neurology and Psychiatry (NCNP)by perforant path stimulation and L-glutamate application, Brainof the Ministry of Health and Welfare, Japan. The authorsRes. 407 (1987) 168–172.

are grateful to Hamamatsu Photonics K.K. for allowing the [10] T. Momiyama, K. Ishihara, T. Serikawa, K. Moritake, M. Sasa,use of the Aqua Cosmos/RATIO Imaging System, digital Effect of nicardipine on abnormal excitability of CA3 pyramidalfluorescence analyzer. cells in hippocampal slices of spontaneously epileptic rats, Eur. J.

Pharmacol. 280 (1995) 119–123.[11] M. Msghina, A.G. Millar, M.P. Charlton, C.K. Govind, H.L.

Atwood, Calcium entry related to active zones and differences inReferences transmitter release at phasic and tonic synapses, J. Neurosci. 19

(1999) 8419–8434.[1] H. Amano, T. Amano, H. Matsubayashi, K. Ishihara, T. Serikawa, [12] M. Sasa, Y. Ohno, H. Ujihara, Y. Fujita, M. Yoshimura, S. Takaori,

M. Sasa, Enhanced calcium influx in hippocampal CA3 neurons of T. Serikawa, J. Yamada, Effects of antiepileptic drugs on absence-spontaneously epileptic rats, Epilepsia 42 (2001) 345–350. like and tonic seizures in the spontaneously epileptic rat, a double

[2] T. Amano, H. Matsubayashi, Y. Fujita, T. Serikawa, H. Tsuru, M. mutant rat, Epilepsia 29 (1988) 505–513.Sasa, J. Yamada, Antiepileptic effects of a newly synthesized Ca [13] T. Serikawa, Y. Ohno, M. Sasa, J. Yamada, S. Takaori, A new modelantagonist, S-312-d, in spontaneously epileptic rats (SER), Jpn. J. of petit mal epilepsy: spontaneous spike and wave discharges inPharmacol. 61 (Suppl. I) (1993) 281. tremor rats, Lab. Anim. 21 (1987) 68–71.

[3] G.E. Barrantes, C.T. Murphy, J. Westwick, S. Wonnacott, Nicotine [14] T. Serikawa, J. Yamada, Epileptic seizures in rats homozygous forincreases intracellular calcium in rat hippocampal neurons via two mutations, zitter and tremor, J. Hered. 77 (1986) 441–444.voltage-gated calcium channels, Neurosci. Lett. 196 (1995) 101– [15] V. Silei, C. Fabrizi, G. Venturini, F. Tagliavini, M. Salmona, O.104. Bugiani, G.M. Lauro, Measurement of intracellular calcium levels

21[4] A.C. Bragdon, D.M. Taylor, J.O. McNamara, W.A. Wilson, Abnor- by the fluorescent Ca indicator Calcium-Green, Brain Res. Protoc.mal hyperexcitability of hippocampal slices from kindled rats is 5 (2000) 132–134.transient, Brain Res. 453 (1988) 257–264. [16] J.L. Stringer, E.W. Lothman, Hippocampal slices from kindled rats

[5] R.P. Haugland, Fura and Indo Ratiometric Calcium Indicators. show an increased sensitivity for induction of epileptiform activityHandbook of Fluorescent Probes and Research Chemicals, Molecu- by changes in extracellular ion concentrations, Neurosci. Lett. 89lar Probes, USA, 2000. (1988) 43–48.

[6] A.M. Hofer, T.E. Machen, Direct measurement of free Ca in [17] A. Takahashi, P. Camacho, J.D. Lechleiter, B. Herman, Measure-organelles of gastric epithelial cells, Am. J. Physiol. 267 (1994) ment of intracellular calcium, Physiol. Rev. 79 (1999) 1089–1125.G442–G451. [18] J. Yamada, T. Serikawa, J. Ishiko, T. Inui, H. Takada, Y. Kawai, A.

[7] K. Ishihara, M. Sasa, T. Momiyama, H. Ujihara, J. Nakamura, T. Okaniwa, Rats with congenital tremor and curled whiskers and hair,Serikawa, J. Yamada, S. Takaori, Abnormal excitability of hip- Jikken Dobutsu 34 (1985) 183–188.