Excitatory and inhibitory pathways modulate kainate excitotoxicity in hippocampal slice cultures

Neuroscience Letters, 154 (1993) 5-8 Elsevier Scientific Publishers Ireland Ltd.

5

NSL 09466

Excitatory and inhibitory pathways modulate kainate excitotoxicity in hippocampal slice cultures Patrizia Casaccia-Bonnefil b, Eirikur Benedikz a, R a b i n d r a Rai a and Peter J. Bergold a'b aDepartment of Pharmacology and bProgram of Anatomy and Cell Biology, SUN Y-HSCB, Brooklyn, N Y 11203 (USA) (Received 22 November 1992; Revised version received 22 January 1993; Accepted 25 January 1993)

Key words. Mossy fiber; Organotypic; N-Methyl-D-aspartate; Kainate; Picrotoxin; Hippocampus In organotypic hippocampal slice cultures, kainate (KA) specifically induces cell loss in the CA3 region while N-methyl-D-aspartate induces cell loss in the CA 1 region. The sensitivity of slice cultures to KA toxicity appears only after 2 weeks in vitro which parallels the appearance of mossy fibers. KA toxicity is potentiated by co-application with the GABA-A antagonist, picrotoxin. These data suggest that the excitotoxicity of KA in slice cultures is modulated by both excitatory and inhibitory synapses.

The CA3 region of the hippocampus is often damaged in patients with severe forms of epilepsy [5,19]. CA3 cell loss also occurs in animals injected with kainate (KA), an experimental model of epilepsy [2,16,21,23]. If mossy fibers are lesioned prior to injection of KA, CA3 pyramidal cells are protected, suggesting that the toxic effect of KA depends on the presence of mossy fiber-CA3 synapses [23,25]. Furthermore, disruption of physiological synaptic connections in the preparation of dissociated neuronal cultures decreases the sensitivity of the cultured cells to KA [13,14]. Organotypic hippocampal slice cultures, in contrast, preserve many of the excitatory and inhibitory synapses of the transverse hippocampal slice [6,10,28,32,33,36]. KA induces cell loss in hippocampal slice cultures in a dose-dependent manner [26,35]. Low concentrations of KA result in a specific loss of CA3 neurons while high concentrations result in complete loss of neurons. Therefore, we have used hippocampal slice cultures to investigate the role of excitatory and inhibitory hippocampal pathways in KA toxicity. Transverse slices (400/~m) are cut using a Mcllwain tissue chopper (Brinkman, Westbury, NY) from the hippocampus of post-natal day 10 Sprague-Dawley rats. The slices are cultured in 50% Eagle's basal media, 25% Earle's balanced salt solution, 25% horse serum, 10 mM Hepes, pH 7.4, 1 mM glutamine and 6.5 mg/ml glucose (all media components are from GIBCO, Grand Island, Correspondence: P.J. Bergold, Department of Pharmacology, Box 29, SUNY-HSCB, Brooklyn, NY 11203, USA. Fax: (1) (718) 2702214.

NY) at 35°C in roller tubes or on transparent membranes (Millicell-CM, Millipore, Bedford, MA) [8,31]. After 4-5 days, glia and other actively dividing cells in the slice cultures are eliminated by treatment with 100 nM 5-flurodeoxyuridine, 100 nM uridine and 100 nM cytosine arabinoside (Sigma, St Louis, MO) for 24 h. After 3 weeks in vitro, the 85% of the cultures retaining the tissue architecture of the transverse hippocampal slice are included in the study. Cultures are incubated for 24 h in medium containing either KA, N-methyl-D-aspartate (NMDA) or picrotoxin (PTX) (Sigma, St Louis, MO), washed and maintained for 1-3 additional days. After treatment, cultures are fixed in 95% ethanol, 5% acetic acid and stained with 1% methylene blue. The roller tube cultures are cleared in xylene and mounted on glass slides using Permount (Fisher Scientific, Pittsburgh, PA). The absence of >90% of neurons in the dentate gyrus, CA3 or CA 1 hippocampal regions is scored as complete loss of neurons in that region. Each slice culture is scored by two independent observers. For Timm's stain, membrane cultures are placed in freshly made 1% sodium sulphide solution for 10 min and then fixed for 15 min in 10% (v/v) formaldehyde (Fisher Scientific, Pittsburgh, PA) in phosphate buffer, pH 7.4. Staining is performed according to the method of Sloviter [29]. Treatment of cultures with KA (100 pM) for 24 h resuits in a consistant and complete loss of neurons (n = 5). KA (5 pM) is selectively toxic to CA3 neurons in slice cultures while KA (1 /.tM) is not toxic (Fig. 1). These results are consistent with previous reports [35,26]. High

Fig. I. Region-specificcell loss induced by KA or NMDA in organotypic hippocampalslicecultures. Slicecultures prepared usingthe roller tube method are untreated (A), treated with 5 ,uM KA (B) or 10 yM NMDA (C) for 24 h and stained with 1% methyleneblue. KA induces loss of CA3 neurons117= 5), while NMDA induces loss of CA1 neurons (n-3). No loss of neurons is seen in control cultures (n = 10). Magnification x2(I.5.

concentrations of N M D A (100 ~M) for 24 h also induces generalized neuronal loss. N M D A (10/IM) selectively kills CAI neurons and N M D A (1 jIM) has no apparent toxic effect (Fig. 1). The specific neuronal loss induced by KA or N M D A correlates with the high density of KAbinding sites in CA3 and of N MDA-binding sites in CA I [18,19]. Mock-treated cultures show no neuronal loss (n = 10). This suggests that the distribution of specific subtypes of glutamate receptors is one determinant of the differential vulnerability of hippocampal neurons to glutamate agonists. Mossy fiber synapses are necessary for CA3 toxicity induced by KA in vivo [22,24,25,34]. Therefore, we studied the development of mossy fibers and their influence on KA toxicity in slice cultures. Mossy fiber terminals are visualized using the Timm sulphide silver method

[29]. Timm staining in stratum lucidum, the region of mossy fiber-CA3 synapses, is not evident in rats until post-natal day 12 [37]. Slice cultures are derived from 10-day-old animals and, therefore, lack Timm staining (Fig. 2). Staining is not observed in cultures maintained in vitro for 7 days, yet is detected in cultures maintained for 21 days (Fig. 2). We and others have shown that the mossy fibers develop during the 2nd week in vitro [6,36]. The development of mossy fibers parallels the appearance of KA toxicity. Cultures maintained for 7 days are resistant to treatment with 5 y M KA (n = 5, data not shown); in contrast, cuttures maintained lbr 3 weeks show CA3 neuronal loss (Fig. 1). These data suggest a role tbr mossy fiber-CA3 synapses in KA toxicity. An alternative explanation is that cultures maintained in vitro for 7 days have a higher threshold for KA toxicity. This is unlikely since the KA receptors which mediate KA toxicity are present in neonatal hippocampi which we use to prepare slice cultures [4,17,18,19]. Inhibitory synapses may also modulate KA toxicity. Inhibitory interneurons are present in organotypic slice cultures [9,10.11,12,28.32,33]. We and others have detected interneurons in these cultures by immunostaining with antibodies against GABA, parvalbumin, calbindin, neuropeptide Y, somatostatin and vasoactive intestinal peptide (P. Casaccia-Bonnefil, R. Rai and P.J. Bergold, unpubl, data) [6,7]. The role of inhibitory neurotransmission in KA toxicity is investigated by co-application of KA with the GABA-A antagonist, PTX. The synergism between PTX and KA can be investigated in slice culture permitting potential enhancement of seizure activity without systemic complications. Treatment of slice cultures with PTX results in interictal spikes and paroxysmal depolarization shifts similar to those observed in the hyperexcitable brain tissue (P. Casaccia-Bonnefil, R. Rai, P.J. Bergold and A. Stelzer, unpubl, data) [32]. Treatment with PTX (50 500/IM) does not induce loss of CA3 pyramidal cells although it produces some loss of granule cells in the dentate gyrus (Fig. 3). Co-application of PTX (50/IM) and KA (1/zM), at doses which are not toxic to CA3 cells when applied individually, results in complete loss of neurons in the hilus, CA3 and the dentate gyrus (Fig. 3). The loss of granule cells induced by co-application of KA and PTX may be due to supragranular sprouting of the mossy fibers as described by Gfihwiler and Zimmer [36]. In vivo suppression of seizure activity protects from KA toxicity [1,15,30]. Our results also suggest that synaptic inhibition mediates a protective action against KA excitotoxicity. Excitatory and inhibitory synapses modulate KA toxicity. PTX may potentiate KA toxicity by enhancing depolarization of CA3 pyramidal cells or by increasing glu-

A

Fig. 2. Development of mossy fibers in organotypic slice cultures. Hippocampal slice cultures prepared using the membrane method from 10-day-old rats are maintained in vitro for 7 days (A) or 21 days (B) and stained using the Timm sulphide silver method [26]. No staining is observed at 7 days (n = 5); at 21 days, staining is seen in the hilus and in CA3, indicating the presence of mature mossy fiber-CA3 synapses in vitro (n = 7). The slice cultures maintained for 7 days are thicker than the cultures at 2l days and appear darker since they contain dead cells as result of antimitotic treatment 2 days earlier. Magnification x20.5.

t a m a t e release b y m o s s y fibers [2,3,27]. K A - b i n d i n g sites

p l i c a t i o n o f K A s t i m u l a t e s the m o s s y f i b e r - C A 3 s y n a p s e s

h a v e b e e n l o c a l i z e d o n m o s s y fiber t e r m i n a l s a n d o n the

b o t h p o s t - a n d p r e - s y n a p t i c a l l y . W h e n the m o s s y fibers

s o m a o f C A 3 p y r a m i d a l cells [18,19]; t h e r e f o r e , b a t h ap-

are i m m a t u r e o r a b s e n t , K A acts o n l y p o s t - s y n a p t i c a l l y o n the C A 3 p y r a m i d a l cells. T h e r e s u l t i n g d e p o l a r i z a t i o n is n o t sufficient to i n d u c e t o x i c i t y [3,16,27]. In c o n t r a s t , the i r r e v e r s i b l e d e p o l a r i z a t i o n i n d u c e d b y K A in the p r e s e n c e o f m a t u r e m o s s y f i b e r - C A 3 s y n a p s e s m a y be sufficient to i n d u c e n e u r o n a l d e a t h [27]. W e t h a n k R. S l o v i t e r f o r a s s i s t a n c e w i t h t h e T i m m s u l p h i d e s t a i n i n g , M . A . Q . S i d d i q u i f o r the g e n e r o u s use o f the tissue c u l t u r e facilities a n d T. S a c k t o r a n d A. Stelzer f o r r e a d i n g this m a n u s c r i p t . T h i s w o r k is s u p p o r t e d by f u n d s f r o m S U N Y - H S C B

a n d the E p i l e p s y F o u n d a -

t i o n o f A m e r i c a . P. C a s a c c i a - B o n n e f i l is a r e c i p i e n t o f the SUNY-HSCB

Fig. 3. KA toxicity is enhanced by decreased synaptic inhibition. Slice cultures prepared using the roller tube method are treated with 1/IM KA (A), 50//M PTX (B) or 1//M KA plus 50/.tM PTX (C) and stained with methylene blue. When I uM KA (n = 5) or 50/IM PTX (n = 5) are applied individually, no toxic effect on CA3 neurons is observed. Coapplication of 1 ,uM KA plus 50 ¢tM PTX results in a complete loss of neurons in CA3 and the dentate gyrus (n = 5). Magnification x20.5.

Competitive Fellowship.

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