Neurotox Res DOI 10.1007/s12640-013-9450-z
ORIGINAL ARTICLE
Enhanced Mossy Fiber Sprouting and Synapse Formation in Organotypic Hippocampal Cultures Following Transient Domoic Acid Excitotoxicity Anabel Pe´rez-Go´mez • R. Andrew Tasker
Received: 25 July 2013 / Revised: 3 December 2013 / Accepted: 5 December 2013 Ó Springer Science+Business Media New York 2013
Abstract We have previously reported evidence of BDNF upregulation and increased neurogenesis in rat organotypic hippocampal slice cultures (OHSC) after a transient excitotoxic injury to the hippocampal CA1 area induced by low concentrations of the AMPA/kainate receptor agonist domoic acid (DOM). The changes observed in OHSC were consistent with observations in vivo, where low concentrations of DOM administered to rats during perinatal development caused increased BDNF and TrkB expression in the resulting adult animals. The in vivo low dose-DOM treatment also results in permanent alterations in hippocampal structure and function, including abnormal formation of dentate granule cell axons projecting to area CA3 (mossy fiber sprouting). Our objective in the current study is to determine if low concentrations of DOM induce mossy fiber sprouting and/or synaptogenesis in OHSC in order to facilitate future studies on the mechanisms of structural hippocampal plasticity induced by DOM. We report herein that application of a low concentration of DOM (2 lM) for 24 h followed by recovery induced a significant increase in the expression of the mossy fiber marker ZnT3 that progressed over time in culture. The DOM insult (2 lM, 24 h) also resulted in a significant upregulation of both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. All of the observed effects were fully antagonized by co-administration of the AMPA/kainate antagonists CNQX or NBQX but only partly by the NMDA antagonist CPP and not by the calcium channel blocker nifedipine. We
A. Pe´rez-Go´mez R. A. Tasker (&) Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada e-mail: [email protected]
conclude that exposure of OHSC to concentrations of DOM below those required to induce permanent neurotoxicity can induce a progressive change in hippocampal structure that can effectively model DOM effects in vivo. Keywords Synaptogenesis Mossy fiber sprouting Glutamate receptors Hippocampal plasticity Epilepsy
Introduction The excitatory amino acid domoic acid (DOM) is a naturally occurring marine toxin analog of kainic acid that acts through glutamate receptors and elicits a very rapid neurotoxic response. In high concentrations, DOM is a potent excitotoxin in vitro (Novelli et al. 1992; Berman and Murray 1997; Sari and Kerr 2001; Jakobsen et al. 2002; Perez-Gomez et al. 2010) and produces dose-dependent neurotoxicity in a variety of species, including humans (Perl et al. 1990; Teitelbaum et al. 1990; Tryphonas et al. 1990a, b; Tasker et al. 1991). The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM-induced toxicity (Gill et al. 2010; Strain and Tasker 1991) and, at lower doses, to DOM-induced structural plasticity relevant to temporal lobe epilepsy (Doucette et al. 2004; Bernard et al. 2007). One of the best characterized forms of hippocampal plasticity, particularly in relation to epilepsy and related seizure disorders, is the abnormal growth and connectivity of dentate granule cell axons [mossy fiber sprouting (MFS)] such that new synaptic connections are formed within the inner molecular layer of dentate gyrus and with basilar dendrites of the pyramidal cells in area CA3 (for reviews see Ben-Ari and Represa 1990; Holmes et al. 2002; Maru et al. 2002). Both axonal
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growth and synaptic plasticity rely on many factors, including the interaction between neurotrophin ligands and their receptors. Brain-derived neurotrophic factor (BDNF) and its high affinity receptor, the tropomyosinrelated kinase B (TrkB), have been reported to have acute and long-term effects on synaptic transmission (Tyler and Pozzo-Miller 2001), synapse number (PozzoMiller et al. 1999) and on the levels of hippocampal synapse proteins (Tartaglia et al. 2001). It is widely reported that neurotrophins (such as BDNF) and their receptors, exhibit altered expression in animal models of epilepsy, as well as in clinical epilepsy (Gall and Isackson 1989; Ernfors et al. 1991; Isackson et al. 1991; Bengzon et al. 1993; Mathern et al. 1996), and these changes may be responsible for the abnormal axonal growth detected in epileptic tissue (Van der Zee et al. 1995; Adams et al. 1997; Binder et al. 1999; Reibel et al. 2000; Lahteinen et al. 2002; Xu et al. 2002). Some studies reveal that different forms of excitatory cellular stimulation can enhance BDNF synthesis and secretion (Nakajima et al. 2001, 2002; Zafra et al. 1992) and, accordingly, we have previously demonstrated that application of a low concentration of DOM followed by recovery stimulates BDNF and TrkB overexpression in rat hippocampal slices in culture (Perez-Gomez and Tasker 2013). The changes observed in organotypic hippocampal slice cultures (OHSC) are consistent with previous in vivo observations from our laboratory in which low doses of DOM administered systemically during postnatal development induced significant increases in hippocampal BDNF and TrkB expression in the resulting adult animals (Doucette et al. 2004; Bernard et al. 2007). The same in vivo treatment also induced changes in hippocampal morphology including significant increases in MFS in various hippocampal subfields. Although primarily studied in vivo, MFS can also be induced in vitro by exposing OHSC to high concentrations of the excitotoxin kainic acid (Routbort et al. 1999; Bausch. 2006). OHSC have been widely used for the investigation of neuronal damage, neuroprotection, and synaptic transmission (Noraberg et al. 1999; Robert et al. 2002; Pringle et al. 2003; Mulholland et al. 2004) allowing the mechanistic and ethical advantages of cell culture to be applied to a developing and fully integrated system. Furthermore, OHSC are well-established, stable model for investigating hippocampal function including developmental synaptogenesis, because neurons maintain synaptogenic ability in each region (CA1, CA3, and DG) (Qin et al. 2001; Mizuhashi et al. 2001; De Simoni et al. 2003). Our objective in the current study was to determine if low concentrations of DOM induce MFS and/or synaptogenesis in OHSC by selective activation of AMPA/kainate receptors in order to facilitate future studies on the
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mechanisms of structural hippocampal plasticity induced by DOM.
Methods Organotypic Hippocampal Slice Cultures (OHSC) OHSC were prepared from 5- to 6-day-old Sprague– Dawley rats of both sexes (Charles River, Quebec, Canada) according to the interface method of Stoppini et al. (1991). Pups were decapitated, the brain was removed, hippocampi were dissected and transversely sliced at a thickness of 400 lm, and transferred into ice-cold dissection buffer containing 1 % penicillin–streptomycin solution (Gibco, NY, USA), 25 mM HEPES (Fisher Scientific, NJ, USA), and 10 mM Tris (Fisher Scientific) in Minimum Essential Medium (Gibco), and selected for clear hippocampal morphology (intact CA regions and dentate gyrus). The slices were transferred onto 0.4-lm porous Millicell membrane inserts (Millipore, MA, USA) and placed in individual 35-mm plates with 1 ml of serum-based medium containing 50 % Minimum Essential Medium (Gibco), 25 % Hanks’ balanced salt solution (Gibco), 12 mM HEPES, 25 % heat-inactivated horse serum (Gibco), and 1 % penicillin–streptomycin solution in a humidified chamber with 5 % CO2 at 37 °C. Media was changed twice a week. All animal care and use procedures were approved in advance by the University of Prince Edward Island Animal Care Committee and were in accordance with the Canadian Council on Animal Care guidelines. All possible efforts were made to minimize animal suffering and the number of animals used. DOM-Induced Excitotoxic Injury and Pharmacological Treatments At 13 days in vitro, damaged OHSC were excluded by propidium iodide (PI) staining (5 lg/ml for 30 min) (PI, Sigma-Aldrich, MO, USA) using a Fluoroarc exciter lamp with a Zeiss Axioplan2 microscope (Carl Zeiss Canada Ltd, ON, Canada). PI-negative slices were exposed to the indicated treatments. Cultures were exposed to DOM (2 lM, BioVectra dcl, Canada) for 24 h and then transferred to a DOM-free medium. The AMPA/kainate receptor (AMPA/KA-R) antagonists NBQX and CNQX (20 lM, Sigma), the NMDA-R antagonist CPP (30 lM, Sigma) or the L-type Ca2? channel blocker nifedipine (NIF, 20 lM, Sigma) were added to the culture medium 15 min before DOM. A minimum of 12 slices from three different preparations were analyzed per treatment. Mean fluorescence was measured in the CA1, CA3, and DG regions, and PI uptake, as an index of neurodegeneration, was defined as
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the ratio of PI fluorescence intensity after treatments to the maximal value obtained in the same slice (designated as 100 %) after low-temperature exposure [4 °C for 24 h immersed in phosphate-buffered saline (PBS)]. Immunohistochemistry Cultures were washed in 0.1 M PBS, treated with 1 % sodium sulfide for 2 h, fixed in formalin for 6 h, dehydrated in 70 % ethanol for 2 h and stored at -20 °C. Sections were washed with PBS for 10 min, placed in 3 % hydrogen peroxide (diluted in PBS) for 20 min, immersed in PBS, and then covered with blocking serum (5.0 % normal goat serum in PBS ? 1 % Triton) for 1 h. The sections were incubated overnight at 4 °C with rabbit antiZnt3 (1:200, Synaptic Systems, Germany #194003) diluted in PBS ? 1 % Triton (PBS-T). After washing with PBS, sections were incubated with biotinylated anti-rabbit IgG (Vector Laboratories, ON, Canada) diluted 1:250 in PBS-T at room temperature for 1 h, then covered with reagent (Vectastain ABC kit, Vector Laboratories) and incubated at room temperature for 30 min. After washing with PBS, bound antibody was visualized by treatment with 2,4-diaminobutyric acid (DAB Staining kit, Vector Laboratories). Finally, the sections were dehydrated in serial ethanol solutions, cleared, and coverslipped in Permount (Fisher Scientific). Negative controls (i.e. buffer substitution) for the primary and secondary antibodies were included in every run and displayed no specific staining at any time. Images were quantified by two experimentally-blind investigators using a 3 point nominal scale (0 = no damage; 1 = moderate, 2 = severe) in all CA subfields and area dentata using a Zeiss Axioplan2 microscope and Axiovision Software. Western Blotting Slice cultures were collected and homogenized on ice in a lysis buffer (63 mM Tris–HCl, 10 % SDS, 2 mM EDTA) and complete protease inhibitor mix (Roche Diagnostics, IN, USA). Protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, IL, USA). Samples were heated to 95 °C for 5 min, and equal quantities of protein extract were separated on 10 % SDS gels. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories Inc., CA, USA, #1620177) and incubated with rabbit anti-ZnT3 (1:700, Synaptic Systems), rabbit antiPSD-95 (1:500, Abcam #AB18258), or mouse anti-SYP (1:1,000, Millipore #MAB368). A control for protein loading was performed by reprobing membranes with an antibody against b-actin or a-tubulin (1:5,000, SigmaAldrich). Membranes were incubated with secondary
anti-mouse or anti-rabbit IgG peroxidase (Sigma-Aldrich, 1:10,000). Immunopositive bands were visualized using the enhanced chemiluminescence (ECL) plus Western blotting system from Amersham (UK). Pictures of the bands were taken, and a subsequent analysis was performed on a Biospectrum AC Imaging System (UVP, Upland, CA, USA) using VisionworksLS software (v 6.7.4, UVP, Upland). Statistical Analysis All data are given as mean ± SEM and statistical significance was evaluated by one-way ANOVA for each hippocampal subfield investigated followed by Tukey’s post hoc test using GraphPad Prism v. 5.0. P \ 0.05 was used as a limit for statistical significance.
Results Effects of Glutamate Receptor Antagonists on DOM Toxicity On day 13 in vitro (DIV) OHSC were exposed to 2 lM DOM for a period of 24 h. In order to quantify the resulting injury in the CA1, CA3 and DG subfields, slices were viewed under a fluorescence microscope to analyze PI uptake and cell death (neuronal and glial) was expressed as a percentage of maximal PI fluorescence (see ‘‘Methods’’ section). As we described previously (Perez-Gomez and Tasker 2012), PI fluorescence intensity in the CA1 subfield reached 25 ± 2 % (n = 12 slices, P \ 0.01, Fig. 1b) in cultures exposed to 2 lM DOM for 24 h, but was not significantly increased in either the CA3 (10 ± 2 %, n = 13 slices, results not shown) or DG (12 ± 4 %, n = 15 slices, results not shown) subfields. Pre-exposure of slices to the AMPA/KA-R antagonist NBQX (20 lM, 15 min) fully prevented the toxic effect of DOM in CA1 (Fig. 1) and PI uptake (6 ± 1 %, n = 12 slices) was comparable to untreated controls (6 ± 1 %, n = 14 slices). Similar results were obtained by pretreating the cells with CNQX (20 lM, 15 min), another AMPA/KA-R antagonist that reduced the toxic effect of 2 lM DOM in CA1 area to control values (5 ± 1 %, n = 15 slices). When combined with DOM, the NMDA-R antagonist CPP (30 lM, 15 min) significantly decreased PI uptake (30 % decrease) compared to that elicited by DOM alone but was not as efficacious as NBQX or CNQX. On the other hand, when coincubated with DOM, nifedipine (NIF, 20 lM, 15 min), a well-known L-type Ca2? channel blocker, failed to reduce DOM toxicity in CA1 subfield (Fig. 1). Because no significant cell death was observed in regions CA3 or DG (see above) results for those regions are not reported.
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Neurotox Res Fig. 1 AMPA/KA-R antagonists fully prevent DOMinduced injury in OHSC. a Fluorescence photomicrographs of PI uptake following 24 h-exposure to the indicated drugs. NBQX (20 lM), CNQX (20 lM), CPP (30 lM), or NIF (20 lM) were added 15 min before DOM (2 lM). b Quantification of PI uptake in the CA1 subfield showed a significant increase in fluorescence after 24 h of incubation with 2 lM DOM. The toxicity is prevented by NBQX and CNQX and significantly decreased by CPP (*P \ 0.01 vs. control alone; #P \ 0.01 vs. ?DOM 24 h alone)
DOM Induces Overexpression of ZnT3 To determine if a low concentration of DOM would induce MFS in OHSC, we measured expression of the zinc transporter 3 (ZnT3) which is abundant in mossy fiber terminals (Palmiter et al. 1996; Chi et al. 2008). OHSC were treated as described previously (Perez-Gomez and Tasker 2012, 2013) in that preparations were exposed to 2 lM DOM for 24 h (insult), changed to a DOM-free medium and subjected to immunohistochemical analysis 14 days post-insult (DPI, see Fig. 2a for time-line). DOM treatment induced a marked increase in ZnT3 labeling at 14 DPI compare to age-matched control slices (Fig. 2b) in the CA3 region but not in CA1 or DG (data not shown). To better examine and quantify the time-course effect of lowdose DOM on MFS plasticity in OHSC, we harvested cultures at three time points following exposure to 2 lM DOM and analyzed the protein samples by Western blotting using the same ZnT3 antibody (see Fig. 2a for timeline). As shown in Fig. 2c, expression of the ZnT3 protein
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was not significantly changed relative to control 3 days post insult (DPI) but DOM treatment did induce a significant increase in ZnT3 expression at 7 DPI (*twofold) compared to age-matched control slices, remaining elevated until the end of the experiment (14 DPI, Fig. 2c). The effects of AMPA/KA-R and NMDA-R antagonists on the DOM-induced increase in ZnT3 expression are shown in Fig. 2d. The AMPA/KA-R antagonists NBQX or CNQX (20 lM, 15 min) both blocked the DOM-stimulated increase in ZnT3 expression in OHSC. In contrast, Western blot analysis demonstrated that both the NMDA-R antagonist CPP and the Ca2? channel blocker NIF failed to suppress or significantly reduce the increased expression of ZnT3 induced by transient injury. Effects of DOM Insult on Pre- and Postsynaptic Protein Expression in Cultured Hippocampal Slices Next, we aimed to determine whether short-term treatment with DOM also elicits long-term biochemical changes in
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Fig. 2 ZnT3 expression is increased after DOM insult. a Experimental design and timeline: 13 DIV slices were treated with 2 lM DOM for 24 h (insult) and then changed to a DOM-free medium. At 0, 3, 7, and 14 DPI lysates for Western blot analysis were collected. At 14 DPI cultures were fixed and subjected to an anti-ZnT3 immunoprotocol. b Representative phase-contrast photomicrographs of ZnT3 labeling of the CA3 subfield. DOM insult resulted in a marked increase in the intensity of ZnT3 immunostaining compared to control. c Effects of transient DOM exposure on ZnT3 expression at different time points post-insult as measured by Western blotting and quantified by densitometric analysis. Values obtained were
normalized to those of b-actin and expressed as the ratio obtained under control conditions from age-matched cultures. Levels of ZnT3 significantly increased with the ongoing incubation in the DOM-free medium (n = 5, *P \ 0.001 vs. control). d DOM-stimulated increases in ZnT3 expression are prevented with AMPA/KA-R antagonists as assessed by Western blotting and quantified by densitometric analysis. The values obtained were normalized to those of b-actin and expressed as the ratio obtained under control conditions from age-matched cultures (n = 3, *P \ 0.001 vs. control alone; #P \ 0.002 vs. ?DOM alone) scale bar 40 lm
hippocampal synapses consistent with synaptogenesis. Detection of variations in synaptic protein concentrations was established using the presynaptic and postsynaptic markers synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95), respectively. Preparations were exposed to 2 lM DOM for 24 h (insult), changed to a DOM-free medium and subjected to immunoblot analysis 14 DPI (see Fig. 3a for time-line). DOM treatment induced a significant increase in SYP expression at 14 DPI compared to age-matched control slices (*1.5-fold, Fig, 3b). We then studied the effects of glutamate receptor antagonists on this DOM-induced increase in SYP expression. Both of the AMPA/KA-R antagonists NBQX and CNQX blocked the overexpression of SYP in the slice cultures. On the other hand, both the NMDA-R antagonist CPP and the Ca2? channel blocker NIF failed to significantly antagonize the increased levels of SYP induced by DOM insult. We then used a similar approach to examine the effect of the DOM-insult on the expression of PSD95. The results obtained are shown in Fig. 3c. Expression of the PSD-95
protein was significantly elevated 14 days after DOM (*1.5-fold). This increase was fully prevented by both NBQX and CNQX. Hippocampal slices incubated with CPP or NIF had PSD-95 concentrations that were not significantly lower than those measured by DOM alone.
Discussion In previous studies, we have demonstrated that a mild reversible injury to OHSC induced by a low concentration of DOM upregulates the neurotrophin BDNF and its corresponding TrkB receptor expression and increases neurogenesis in both the dentate gyrus and CA1 subfields (Perez-Gomez and Tasker 2012, 2013). The changes observed previously in OHSC were consistent with observations in vivo, where low concentrations of DOM administered to rats during perinatal development caused increased BDNF and TrkB expression in the resulting adult animals (Doucette et al. 2004; Bernard et al. 2007).
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Fig. 3 Effects of DOM insult on presynaptic and postsynaptic marker expression. a Experimental design and timeline: OHSC were treated with 2 lM DOM for 24 h (insult) and transferred to DOM-free medium. At 14 DPI protein samples were collected. The effects of transient DOM exposure on synaptophysin (SYP, b) and PSD-95 (c) expression as investigated by Western blotting. Amounts of PSD95 and SYP were assessed by densitometric analysis, and the values obtained were normalized to those of a-tubulin (SYP) or b-actin (PSD-95) and expressed as the ratio obtained under control conditions from age-matched cultures (n = 3, *P \ 0.002 vs. control alone; #P \ 0.001 vs. ?DOM alone)
Because permanent alterations in hippocampal structure have also been described after the in vivo treatment, in the present study we investigated whether DOM insult induces hippocampal plasticity and remodeling in the organotypic in vitro model. Our results showed that DOM treatment induces AMPA/KA-R dependent MFS and promotes synaptogenesis in OHSC.
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MFS is one of the best characterized examples of structural plasticity in the hippocampus and is considered a defining feature in animal models of epilepsy, including those that use injections of kainic acid to induce convulsive seizures (for review see Holmes et al. 1998, 2002). Presynaptic boutons of the MF contain an exceptionally high concentration of zinc in the synaptic vesicles (Frederickson et al. 2000) which is co-released with glutamate at mossy fiber synapses and then returned to the pre-synaptic terminal by the action of the synaptic-vesicle-specific transporter ZnT3 (Palmiter et al. 1996; Jo et al. 2000; Mitsuya et al. 2009; Wang et al. 2002). Earlier studies reported that the expression of ZnT3 is identical to the pattern of results observed with the classic Timm’s staining method (Palmiter et al. 1996; Wenzel et al. 1997), so ZnT3 has been extensively used as a marker of MF projections (Chi et al. 2008; McGonigal et al. 2012; Rekart and Routtenberg. 2010). Exposure of OHSC to 2 lM DOM for 24 h (insult) followed by recovery induced an overexpression of ZnT3 in the CA3 area, which is the primary projection target for dentate granule cell axons (mossy fibers). Collectively, the previously published in vivo data and the in vitro data reported herein support the notion that DOM induces MFS. As we described before, exposure of OHSC to concentrations of DOM below those required to induce permanent neurotoxicity induce proliferation and differentiation of neural progenitor cells (Perez-Gomez and Tasker 2012), and according to previous studies, the newborn granule cell axons require 10 days to grow and integrate into the CA3 after their generation (Zhao et al. 2006). Because, in the current study, DOM insult induced a significant overexpression in ZnT3 protein 1 week after exposure, additional experiments would be required to quantify the contribution of newly born granule cell axons to the expansion of the MF field. In order to quantify a possible increased synaptogenesis after DOM insult, expression of both the presynaptic marker protein synaptophysin and the postsynaptic protein PSD-95 was measured as the concentration of these two proteins closely correlate with synapse number and are commonly used to assay for loss or gain of synapses (Kelly et al. 2005; Sabbagh et al. 1999; Tiraboschi et al. 2000) and as markers of synaptic activity (Tartaglia et al. 2001; Chavis and Westbrook 2001). Our results show clearly that DOM insult produces a significant overexpression of both SYP and PSD-95, indicating increased synaptogenesis in addition to MFS. Both axonal growth and synaptic plasticity are regulated by neurotrophins and their receptors, and TrkB and BDNF, among others, have been proven to be crucial in the differentiation, neurite outgrowth, synaptic transmission and levels of hippocampal synapse proteins (Tyler and PozzoMiller. 2001; Pozzo-Miller et al. 1999; Tartaglia et al.
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2001; Barde 1989; Lu and Figurov 1997). The results presented above combined with previous published data from our laboratory demonstrating increased hippocampal BDNF and TrkB expression both in vivo (Doucette et al. 2004; Bernard et al. 2007) and in vitro (Perez-Gomez and Tasker 2013) add further support to the argument that alterations in neurotrophin levels may lead to the aberrant axonal growth observed within the adult brain (Van der Zee et al. 1995; Adams et al. 1997; Binder et al. 1999; Reibel et al. 2000; Lahteinen et al. 2002; Xu et al. 2002, 2004). Excitotoxicity is accompanied by an increase in calcium levels in neurons (Vanderklish and Bahr 2000; Mattson 2003). Further, a significant intracellular calcium increase following treatment with DOM has been shown before (Fernandez-Sanchez and Novelli 1996). In the current study, however, the L-type Ca2? channel blocker NIF failed to decrease both neurotoxicity and overexpression of synaptic markers after DOM treatment in OHSC, a result that may suggest that increased calcium concentration does not play a prominent role in the hippocampal changes observed. On the other hand, more experiments would be necessary in order to quantify the efficiency of NIF at decreasing intracellular calcium concentration after DOM treatment in OHSC. To investigate the pharmacology of both DOM-induced neurotoxicity and hippocampal plasticity, we administered various selective antagonists prior to DOM in OHSCs prepared from rat pups of both sexes to be consistent with both our previous work (Perez-Gomez and Tasker 2012, 2013) and that of others (Jung et al. 2013; Inada et al. 2013). Two recent papers (Butler et al. 2009; Walls et al. 2013), however, reported sex differences in toxicity in OHSCs so separation of slices from male and female rats may be prudent in future studies. DOM-induced neurotoxicity in OHSC seemed to be mediated mainly through the AMPA/KA-R, as both of the antagonists NBQX and CNQX completely blocked the increased neurodegeneration observed after 24 h treatment with the excitotoxin. Although not as effective as AMPA/KA-R antagonists, the NMDA-R antagonist CPP significantly reduced the DOMincreased percentage of dead neurons. Earlier studies reported a similar result, showing that although DOM toxicity is mediated primarily by AMPA/KA receptors both in vivo and in vitro (Qiu et al. 2006; Qiu and CurrasCollazo 2006; Renard et al. 1995; Hampson et al. 1992), NMDA receptors are also involved (Novelli et al. 1992; Berman and Murray 1997; Tasker and Strain 1998). AMPA/KA receptors seem to be involved in both pre- and post-synaptic functions in the nervous system, and are important in learning and synaptic plasticity (Jane et al. 2009; Vincent and Mulle 2009), and accordingly, AMPA/ KA-R antagonists prevented DOM-induced MFS and
increased synaptogenesis in OHSC. On the other hand, the NMDA-R antagonist failed to decrease both the ZnT3 overexpression and the increased SYP and PSD-95 levels induced by DOM treatment, possibly because there was still sufficient residual toxicity to stimulate these processes. These results suggest that DOM neurotoxicity in OHSC is in part mediated by NMDA receptors but AMPA/KArelated mechanisms may dominate in the long term hippocampal plastic changes induced by the action of DOM. In summary, our results show clearly that low dose DOM-induced AMPA/KA-dependent hippocampal plasticity, which is well characterized in whole animals, can be effectively modeled in OHSC, thereby permitting future mechanistic investigations of the MFS phenomenon, the associated increase in synaptogenesis and the functional consequences of abnormal hippocampal development. Acknowledgments The authors thank Ms. Emily McGuire for expert technical assistance. This work was funded by Atlantic Innovation Fund Grant 193639. The facilities of the Atlantic Center for Comparative Biomedical Research were made available at no cost to the investigators. Conflict of interest of interest.
The authors declare that they have no conflict
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ORIGINAL ARTICLE
Enhanced Mossy Fiber Sprouting and Synapse Formation in Organotypic Hippocampal Cultures Following Transient Domoic Acid Excitotoxicity Anabel Pe´rez-Go´mez • R. Andrew Tasker
Received: 25 July 2013 / Revised: 3 December 2013 / Accepted: 5 December 2013 Ó Springer Science+Business Media New York 2013
Abstract We have previously reported evidence of BDNF upregulation and increased neurogenesis in rat organotypic hippocampal slice cultures (OHSC) after a transient excitotoxic injury to the hippocampal CA1 area induced by low concentrations of the AMPA/kainate receptor agonist domoic acid (DOM). The changes observed in OHSC were consistent with observations in vivo, where low concentrations of DOM administered to rats during perinatal development caused increased BDNF and TrkB expression in the resulting adult animals. The in vivo low dose-DOM treatment also results in permanent alterations in hippocampal structure and function, including abnormal formation of dentate granule cell axons projecting to area CA3 (mossy fiber sprouting). Our objective in the current study is to determine if low concentrations of DOM induce mossy fiber sprouting and/or synaptogenesis in OHSC in order to facilitate future studies on the mechanisms of structural hippocampal plasticity induced by DOM. We report herein that application of a low concentration of DOM (2 lM) for 24 h followed by recovery induced a significant increase in the expression of the mossy fiber marker ZnT3 that progressed over time in culture. The DOM insult (2 lM, 24 h) also resulted in a significant upregulation of both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. All of the observed effects were fully antagonized by co-administration of the AMPA/kainate antagonists CNQX or NBQX but only partly by the NMDA antagonist CPP and not by the calcium channel blocker nifedipine. We
A. Pe´rez-Go´mez R. A. Tasker (&) Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada e-mail: [email protected]
conclude that exposure of OHSC to concentrations of DOM below those required to induce permanent neurotoxicity can induce a progressive change in hippocampal structure that can effectively model DOM effects in vivo. Keywords Synaptogenesis Mossy fiber sprouting Glutamate receptors Hippocampal plasticity Epilepsy
Introduction The excitatory amino acid domoic acid (DOM) is a naturally occurring marine toxin analog of kainic acid that acts through glutamate receptors and elicits a very rapid neurotoxic response. In high concentrations, DOM is a potent excitotoxin in vitro (Novelli et al. 1992; Berman and Murray 1997; Sari and Kerr 2001; Jakobsen et al. 2002; Perez-Gomez et al. 2010) and produces dose-dependent neurotoxicity in a variety of species, including humans (Perl et al. 1990; Teitelbaum et al. 1990; Tryphonas et al. 1990a, b; Tasker et al. 1991). The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM-induced toxicity (Gill et al. 2010; Strain and Tasker 1991) and, at lower doses, to DOM-induced structural plasticity relevant to temporal lobe epilepsy (Doucette et al. 2004; Bernard et al. 2007). One of the best characterized forms of hippocampal plasticity, particularly in relation to epilepsy and related seizure disorders, is the abnormal growth and connectivity of dentate granule cell axons [mossy fiber sprouting (MFS)] such that new synaptic connections are formed within the inner molecular layer of dentate gyrus and with basilar dendrites of the pyramidal cells in area CA3 (for reviews see Ben-Ari and Represa 1990; Holmes et al. 2002; Maru et al. 2002). Both axonal
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growth and synaptic plasticity rely on many factors, including the interaction between neurotrophin ligands and their receptors. Brain-derived neurotrophic factor (BDNF) and its high affinity receptor, the tropomyosinrelated kinase B (TrkB), have been reported to have acute and long-term effects on synaptic transmission (Tyler and Pozzo-Miller 2001), synapse number (PozzoMiller et al. 1999) and on the levels of hippocampal synapse proteins (Tartaglia et al. 2001). It is widely reported that neurotrophins (such as BDNF) and their receptors, exhibit altered expression in animal models of epilepsy, as well as in clinical epilepsy (Gall and Isackson 1989; Ernfors et al. 1991; Isackson et al. 1991; Bengzon et al. 1993; Mathern et al. 1996), and these changes may be responsible for the abnormal axonal growth detected in epileptic tissue (Van der Zee et al. 1995; Adams et al. 1997; Binder et al. 1999; Reibel et al. 2000; Lahteinen et al. 2002; Xu et al. 2002). Some studies reveal that different forms of excitatory cellular stimulation can enhance BDNF synthesis and secretion (Nakajima et al. 2001, 2002; Zafra et al. 1992) and, accordingly, we have previously demonstrated that application of a low concentration of DOM followed by recovery stimulates BDNF and TrkB overexpression in rat hippocampal slices in culture (Perez-Gomez and Tasker 2013). The changes observed in organotypic hippocampal slice cultures (OHSC) are consistent with previous in vivo observations from our laboratory in which low doses of DOM administered systemically during postnatal development induced significant increases in hippocampal BDNF and TrkB expression in the resulting adult animals (Doucette et al. 2004; Bernard et al. 2007). The same in vivo treatment also induced changes in hippocampal morphology including significant increases in MFS in various hippocampal subfields. Although primarily studied in vivo, MFS can also be induced in vitro by exposing OHSC to high concentrations of the excitotoxin kainic acid (Routbort et al. 1999; Bausch. 2006). OHSC have been widely used for the investigation of neuronal damage, neuroprotection, and synaptic transmission (Noraberg et al. 1999; Robert et al. 2002; Pringle et al. 2003; Mulholland et al. 2004) allowing the mechanistic and ethical advantages of cell culture to be applied to a developing and fully integrated system. Furthermore, OHSC are well-established, stable model for investigating hippocampal function including developmental synaptogenesis, because neurons maintain synaptogenic ability in each region (CA1, CA3, and DG) (Qin et al. 2001; Mizuhashi et al. 2001; De Simoni et al. 2003). Our objective in the current study was to determine if low concentrations of DOM induce MFS and/or synaptogenesis in OHSC by selective activation of AMPA/kainate receptors in order to facilitate future studies on the
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mechanisms of structural hippocampal plasticity induced by DOM.
Methods Organotypic Hippocampal Slice Cultures (OHSC) OHSC were prepared from 5- to 6-day-old Sprague– Dawley rats of both sexes (Charles River, Quebec, Canada) according to the interface method of Stoppini et al. (1991). Pups were decapitated, the brain was removed, hippocampi were dissected and transversely sliced at a thickness of 400 lm, and transferred into ice-cold dissection buffer containing 1 % penicillin–streptomycin solution (Gibco, NY, USA), 25 mM HEPES (Fisher Scientific, NJ, USA), and 10 mM Tris (Fisher Scientific) in Minimum Essential Medium (Gibco), and selected for clear hippocampal morphology (intact CA regions and dentate gyrus). The slices were transferred onto 0.4-lm porous Millicell membrane inserts (Millipore, MA, USA) and placed in individual 35-mm plates with 1 ml of serum-based medium containing 50 % Minimum Essential Medium (Gibco), 25 % Hanks’ balanced salt solution (Gibco), 12 mM HEPES, 25 % heat-inactivated horse serum (Gibco), and 1 % penicillin–streptomycin solution in a humidified chamber with 5 % CO2 at 37 °C. Media was changed twice a week. All animal care and use procedures were approved in advance by the University of Prince Edward Island Animal Care Committee and were in accordance with the Canadian Council on Animal Care guidelines. All possible efforts were made to minimize animal suffering and the number of animals used. DOM-Induced Excitotoxic Injury and Pharmacological Treatments At 13 days in vitro, damaged OHSC were excluded by propidium iodide (PI) staining (5 lg/ml for 30 min) (PI, Sigma-Aldrich, MO, USA) using a Fluoroarc exciter lamp with a Zeiss Axioplan2 microscope (Carl Zeiss Canada Ltd, ON, Canada). PI-negative slices were exposed to the indicated treatments. Cultures were exposed to DOM (2 lM, BioVectra dcl, Canada) for 24 h and then transferred to a DOM-free medium. The AMPA/kainate receptor (AMPA/KA-R) antagonists NBQX and CNQX (20 lM, Sigma), the NMDA-R antagonist CPP (30 lM, Sigma) or the L-type Ca2? channel blocker nifedipine (NIF, 20 lM, Sigma) were added to the culture medium 15 min before DOM. A minimum of 12 slices from three different preparations were analyzed per treatment. Mean fluorescence was measured in the CA1, CA3, and DG regions, and PI uptake, as an index of neurodegeneration, was defined as
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the ratio of PI fluorescence intensity after treatments to the maximal value obtained in the same slice (designated as 100 %) after low-temperature exposure [4 °C for 24 h immersed in phosphate-buffered saline (PBS)]. Immunohistochemistry Cultures were washed in 0.1 M PBS, treated with 1 % sodium sulfide for 2 h, fixed in formalin for 6 h, dehydrated in 70 % ethanol for 2 h and stored at -20 °C. Sections were washed with PBS for 10 min, placed in 3 % hydrogen peroxide (diluted in PBS) for 20 min, immersed in PBS, and then covered with blocking serum (5.0 % normal goat serum in PBS ? 1 % Triton) for 1 h. The sections were incubated overnight at 4 °C with rabbit antiZnt3 (1:200, Synaptic Systems, Germany #194003) diluted in PBS ? 1 % Triton (PBS-T). After washing with PBS, sections were incubated with biotinylated anti-rabbit IgG (Vector Laboratories, ON, Canada) diluted 1:250 in PBS-T at room temperature for 1 h, then covered with reagent (Vectastain ABC kit, Vector Laboratories) and incubated at room temperature for 30 min. After washing with PBS, bound antibody was visualized by treatment with 2,4-diaminobutyric acid (DAB Staining kit, Vector Laboratories). Finally, the sections were dehydrated in serial ethanol solutions, cleared, and coverslipped in Permount (Fisher Scientific). Negative controls (i.e. buffer substitution) for the primary and secondary antibodies were included in every run and displayed no specific staining at any time. Images were quantified by two experimentally-blind investigators using a 3 point nominal scale (0 = no damage; 1 = moderate, 2 = severe) in all CA subfields and area dentata using a Zeiss Axioplan2 microscope and Axiovision Software. Western Blotting Slice cultures were collected and homogenized on ice in a lysis buffer (63 mM Tris–HCl, 10 % SDS, 2 mM EDTA) and complete protease inhibitor mix (Roche Diagnostics, IN, USA). Protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, IL, USA). Samples were heated to 95 °C for 5 min, and equal quantities of protein extract were separated on 10 % SDS gels. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories Inc., CA, USA, #1620177) and incubated with rabbit anti-ZnT3 (1:700, Synaptic Systems), rabbit antiPSD-95 (1:500, Abcam #AB18258), or mouse anti-SYP (1:1,000, Millipore #MAB368). A control for protein loading was performed by reprobing membranes with an antibody against b-actin or a-tubulin (1:5,000, SigmaAldrich). Membranes were incubated with secondary
anti-mouse or anti-rabbit IgG peroxidase (Sigma-Aldrich, 1:10,000). Immunopositive bands were visualized using the enhanced chemiluminescence (ECL) plus Western blotting system from Amersham (UK). Pictures of the bands were taken, and a subsequent analysis was performed on a Biospectrum AC Imaging System (UVP, Upland, CA, USA) using VisionworksLS software (v 6.7.4, UVP, Upland). Statistical Analysis All data are given as mean ± SEM and statistical significance was evaluated by one-way ANOVA for each hippocampal subfield investigated followed by Tukey’s post hoc test using GraphPad Prism v. 5.0. P \ 0.05 was used as a limit for statistical significance.
Results Effects of Glutamate Receptor Antagonists on DOM Toxicity On day 13 in vitro (DIV) OHSC were exposed to 2 lM DOM for a period of 24 h. In order to quantify the resulting injury in the CA1, CA3 and DG subfields, slices were viewed under a fluorescence microscope to analyze PI uptake and cell death (neuronal and glial) was expressed as a percentage of maximal PI fluorescence (see ‘‘Methods’’ section). As we described previously (Perez-Gomez and Tasker 2012), PI fluorescence intensity in the CA1 subfield reached 25 ± 2 % (n = 12 slices, P \ 0.01, Fig. 1b) in cultures exposed to 2 lM DOM for 24 h, but was not significantly increased in either the CA3 (10 ± 2 %, n = 13 slices, results not shown) or DG (12 ± 4 %, n = 15 slices, results not shown) subfields. Pre-exposure of slices to the AMPA/KA-R antagonist NBQX (20 lM, 15 min) fully prevented the toxic effect of DOM in CA1 (Fig. 1) and PI uptake (6 ± 1 %, n = 12 slices) was comparable to untreated controls (6 ± 1 %, n = 14 slices). Similar results were obtained by pretreating the cells with CNQX (20 lM, 15 min), another AMPA/KA-R antagonist that reduced the toxic effect of 2 lM DOM in CA1 area to control values (5 ± 1 %, n = 15 slices). When combined with DOM, the NMDA-R antagonist CPP (30 lM, 15 min) significantly decreased PI uptake (30 % decrease) compared to that elicited by DOM alone but was not as efficacious as NBQX or CNQX. On the other hand, when coincubated with DOM, nifedipine (NIF, 20 lM, 15 min), a well-known L-type Ca2? channel blocker, failed to reduce DOM toxicity in CA1 subfield (Fig. 1). Because no significant cell death was observed in regions CA3 or DG (see above) results for those regions are not reported.
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Neurotox Res Fig. 1 AMPA/KA-R antagonists fully prevent DOMinduced injury in OHSC. a Fluorescence photomicrographs of PI uptake following 24 h-exposure to the indicated drugs. NBQX (20 lM), CNQX (20 lM), CPP (30 lM), or NIF (20 lM) were added 15 min before DOM (2 lM). b Quantification of PI uptake in the CA1 subfield showed a significant increase in fluorescence after 24 h of incubation with 2 lM DOM. The toxicity is prevented by NBQX and CNQX and significantly decreased by CPP (*P \ 0.01 vs. control alone; #P \ 0.01 vs. ?DOM 24 h alone)
DOM Induces Overexpression of ZnT3 To determine if a low concentration of DOM would induce MFS in OHSC, we measured expression of the zinc transporter 3 (ZnT3) which is abundant in mossy fiber terminals (Palmiter et al. 1996; Chi et al. 2008). OHSC were treated as described previously (Perez-Gomez and Tasker 2012, 2013) in that preparations were exposed to 2 lM DOM for 24 h (insult), changed to a DOM-free medium and subjected to immunohistochemical analysis 14 days post-insult (DPI, see Fig. 2a for time-line). DOM treatment induced a marked increase in ZnT3 labeling at 14 DPI compare to age-matched control slices (Fig. 2b) in the CA3 region but not in CA1 or DG (data not shown). To better examine and quantify the time-course effect of lowdose DOM on MFS plasticity in OHSC, we harvested cultures at three time points following exposure to 2 lM DOM and analyzed the protein samples by Western blotting using the same ZnT3 antibody (see Fig. 2a for timeline). As shown in Fig. 2c, expression of the ZnT3 protein
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was not significantly changed relative to control 3 days post insult (DPI) but DOM treatment did induce a significant increase in ZnT3 expression at 7 DPI (*twofold) compared to age-matched control slices, remaining elevated until the end of the experiment (14 DPI, Fig. 2c). The effects of AMPA/KA-R and NMDA-R antagonists on the DOM-induced increase in ZnT3 expression are shown in Fig. 2d. The AMPA/KA-R antagonists NBQX or CNQX (20 lM, 15 min) both blocked the DOM-stimulated increase in ZnT3 expression in OHSC. In contrast, Western blot analysis demonstrated that both the NMDA-R antagonist CPP and the Ca2? channel blocker NIF failed to suppress or significantly reduce the increased expression of ZnT3 induced by transient injury. Effects of DOM Insult on Pre- and Postsynaptic Protein Expression in Cultured Hippocampal Slices Next, we aimed to determine whether short-term treatment with DOM also elicits long-term biochemical changes in
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Fig. 2 ZnT3 expression is increased after DOM insult. a Experimental design and timeline: 13 DIV slices were treated with 2 lM DOM for 24 h (insult) and then changed to a DOM-free medium. At 0, 3, 7, and 14 DPI lysates for Western blot analysis were collected. At 14 DPI cultures were fixed and subjected to an anti-ZnT3 immunoprotocol. b Representative phase-contrast photomicrographs of ZnT3 labeling of the CA3 subfield. DOM insult resulted in a marked increase in the intensity of ZnT3 immunostaining compared to control. c Effects of transient DOM exposure on ZnT3 expression at different time points post-insult as measured by Western blotting and quantified by densitometric analysis. Values obtained were
normalized to those of b-actin and expressed as the ratio obtained under control conditions from age-matched cultures. Levels of ZnT3 significantly increased with the ongoing incubation in the DOM-free medium (n = 5, *P \ 0.001 vs. control). d DOM-stimulated increases in ZnT3 expression are prevented with AMPA/KA-R antagonists as assessed by Western blotting and quantified by densitometric analysis. The values obtained were normalized to those of b-actin and expressed as the ratio obtained under control conditions from age-matched cultures (n = 3, *P \ 0.001 vs. control alone; #P \ 0.002 vs. ?DOM alone) scale bar 40 lm
hippocampal synapses consistent with synaptogenesis. Detection of variations in synaptic protein concentrations was established using the presynaptic and postsynaptic markers synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95), respectively. Preparations were exposed to 2 lM DOM for 24 h (insult), changed to a DOM-free medium and subjected to immunoblot analysis 14 DPI (see Fig. 3a for time-line). DOM treatment induced a significant increase in SYP expression at 14 DPI compared to age-matched control slices (*1.5-fold, Fig, 3b). We then studied the effects of glutamate receptor antagonists on this DOM-induced increase in SYP expression. Both of the AMPA/KA-R antagonists NBQX and CNQX blocked the overexpression of SYP in the slice cultures. On the other hand, both the NMDA-R antagonist CPP and the Ca2? channel blocker NIF failed to significantly antagonize the increased levels of SYP induced by DOM insult. We then used a similar approach to examine the effect of the DOM-insult on the expression of PSD95. The results obtained are shown in Fig. 3c. Expression of the PSD-95
protein was significantly elevated 14 days after DOM (*1.5-fold). This increase was fully prevented by both NBQX and CNQX. Hippocampal slices incubated with CPP or NIF had PSD-95 concentrations that were not significantly lower than those measured by DOM alone.
Discussion In previous studies, we have demonstrated that a mild reversible injury to OHSC induced by a low concentration of DOM upregulates the neurotrophin BDNF and its corresponding TrkB receptor expression and increases neurogenesis in both the dentate gyrus and CA1 subfields (Perez-Gomez and Tasker 2012, 2013). The changes observed previously in OHSC were consistent with observations in vivo, where low concentrations of DOM administered to rats during perinatal development caused increased BDNF and TrkB expression in the resulting adult animals (Doucette et al. 2004; Bernard et al. 2007).
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Fig. 3 Effects of DOM insult on presynaptic and postsynaptic marker expression. a Experimental design and timeline: OHSC were treated with 2 lM DOM for 24 h (insult) and transferred to DOM-free medium. At 14 DPI protein samples were collected. The effects of transient DOM exposure on synaptophysin (SYP, b) and PSD-95 (c) expression as investigated by Western blotting. Amounts of PSD95 and SYP were assessed by densitometric analysis, and the values obtained were normalized to those of a-tubulin (SYP) or b-actin (PSD-95) and expressed as the ratio obtained under control conditions from age-matched cultures (n = 3, *P \ 0.002 vs. control alone; #P \ 0.001 vs. ?DOM alone)
Because permanent alterations in hippocampal structure have also been described after the in vivo treatment, in the present study we investigated whether DOM insult induces hippocampal plasticity and remodeling in the organotypic in vitro model. Our results showed that DOM treatment induces AMPA/KA-R dependent MFS and promotes synaptogenesis in OHSC.
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MFS is one of the best characterized examples of structural plasticity in the hippocampus and is considered a defining feature in animal models of epilepsy, including those that use injections of kainic acid to induce convulsive seizures (for review see Holmes et al. 1998, 2002). Presynaptic boutons of the MF contain an exceptionally high concentration of zinc in the synaptic vesicles (Frederickson et al. 2000) which is co-released with glutamate at mossy fiber synapses and then returned to the pre-synaptic terminal by the action of the synaptic-vesicle-specific transporter ZnT3 (Palmiter et al. 1996; Jo et al. 2000; Mitsuya et al. 2009; Wang et al. 2002). Earlier studies reported that the expression of ZnT3 is identical to the pattern of results observed with the classic Timm’s staining method (Palmiter et al. 1996; Wenzel et al. 1997), so ZnT3 has been extensively used as a marker of MF projections (Chi et al. 2008; McGonigal et al. 2012; Rekart and Routtenberg. 2010). Exposure of OHSC to 2 lM DOM for 24 h (insult) followed by recovery induced an overexpression of ZnT3 in the CA3 area, which is the primary projection target for dentate granule cell axons (mossy fibers). Collectively, the previously published in vivo data and the in vitro data reported herein support the notion that DOM induces MFS. As we described before, exposure of OHSC to concentrations of DOM below those required to induce permanent neurotoxicity induce proliferation and differentiation of neural progenitor cells (Perez-Gomez and Tasker 2012), and according to previous studies, the newborn granule cell axons require 10 days to grow and integrate into the CA3 after their generation (Zhao et al. 2006). Because, in the current study, DOM insult induced a significant overexpression in ZnT3 protein 1 week after exposure, additional experiments would be required to quantify the contribution of newly born granule cell axons to the expansion of the MF field. In order to quantify a possible increased synaptogenesis after DOM insult, expression of both the presynaptic marker protein synaptophysin and the postsynaptic protein PSD-95 was measured as the concentration of these two proteins closely correlate with synapse number and are commonly used to assay for loss or gain of synapses (Kelly et al. 2005; Sabbagh et al. 1999; Tiraboschi et al. 2000) and as markers of synaptic activity (Tartaglia et al. 2001; Chavis and Westbrook 2001). Our results show clearly that DOM insult produces a significant overexpression of both SYP and PSD-95, indicating increased synaptogenesis in addition to MFS. Both axonal growth and synaptic plasticity are regulated by neurotrophins and their receptors, and TrkB and BDNF, among others, have been proven to be crucial in the differentiation, neurite outgrowth, synaptic transmission and levels of hippocampal synapse proteins (Tyler and PozzoMiller. 2001; Pozzo-Miller et al. 1999; Tartaglia et al.
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2001; Barde 1989; Lu and Figurov 1997). The results presented above combined with previous published data from our laboratory demonstrating increased hippocampal BDNF and TrkB expression both in vivo (Doucette et al. 2004; Bernard et al. 2007) and in vitro (Perez-Gomez and Tasker 2013) add further support to the argument that alterations in neurotrophin levels may lead to the aberrant axonal growth observed within the adult brain (Van der Zee et al. 1995; Adams et al. 1997; Binder et al. 1999; Reibel et al. 2000; Lahteinen et al. 2002; Xu et al. 2002, 2004). Excitotoxicity is accompanied by an increase in calcium levels in neurons (Vanderklish and Bahr 2000; Mattson 2003). Further, a significant intracellular calcium increase following treatment with DOM has been shown before (Fernandez-Sanchez and Novelli 1996). In the current study, however, the L-type Ca2? channel blocker NIF failed to decrease both neurotoxicity and overexpression of synaptic markers after DOM treatment in OHSC, a result that may suggest that increased calcium concentration does not play a prominent role in the hippocampal changes observed. On the other hand, more experiments would be necessary in order to quantify the efficiency of NIF at decreasing intracellular calcium concentration after DOM treatment in OHSC. To investigate the pharmacology of both DOM-induced neurotoxicity and hippocampal plasticity, we administered various selective antagonists prior to DOM in OHSCs prepared from rat pups of both sexes to be consistent with both our previous work (Perez-Gomez and Tasker 2012, 2013) and that of others (Jung et al. 2013; Inada et al. 2013). Two recent papers (Butler et al. 2009; Walls et al. 2013), however, reported sex differences in toxicity in OHSCs so separation of slices from male and female rats may be prudent in future studies. DOM-induced neurotoxicity in OHSC seemed to be mediated mainly through the AMPA/KA-R, as both of the antagonists NBQX and CNQX completely blocked the increased neurodegeneration observed after 24 h treatment with the excitotoxin. Although not as effective as AMPA/KA-R antagonists, the NMDA-R antagonist CPP significantly reduced the DOMincreased percentage of dead neurons. Earlier studies reported a similar result, showing that although DOM toxicity is mediated primarily by AMPA/KA receptors both in vivo and in vitro (Qiu et al. 2006; Qiu and CurrasCollazo 2006; Renard et al. 1995; Hampson et al. 1992), NMDA receptors are also involved (Novelli et al. 1992; Berman and Murray 1997; Tasker and Strain 1998). AMPA/KA receptors seem to be involved in both pre- and post-synaptic functions in the nervous system, and are important in learning and synaptic plasticity (Jane et al. 2009; Vincent and Mulle 2009), and accordingly, AMPA/ KA-R antagonists prevented DOM-induced MFS and
increased synaptogenesis in OHSC. On the other hand, the NMDA-R antagonist failed to decrease both the ZnT3 overexpression and the increased SYP and PSD-95 levels induced by DOM treatment, possibly because there was still sufficient residual toxicity to stimulate these processes. These results suggest that DOM neurotoxicity in OHSC is in part mediated by NMDA receptors but AMPA/KArelated mechanisms may dominate in the long term hippocampal plastic changes induced by the action of DOM. In summary, our results show clearly that low dose DOM-induced AMPA/KA-dependent hippocampal plasticity, which is well characterized in whole animals, can be effectively modeled in OHSC, thereby permitting future mechanistic investigations of the MFS phenomenon, the associated increase in synaptogenesis and the functional consequences of abnormal hippocampal development. Acknowledgments The authors thank Ms. Emily McGuire for expert technical assistance. This work was funded by Atlantic Innovation Fund Grant 193639. The facilities of the Atlantic Center for Comparative Biomedical Research were made available at no cost to the investigators. Conflict of interest of interest.
The authors declare that they have no conflict
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