Plasticity and Metaplasticity of Lateral Perforant Path in Hippocampal Dentate Gyrus in a Rat Model of Febrile Seizure

124 Acta Physiologica Sinica, April 25, 2011, 63(2): 124–130 http://www.actaps.com.cn

Research Paper

Plasticity and metaplasticity of lateral perforant path in hippocampal dentate gyrus in a rat model of febrile seizure

ZHANG Lian*, LUO Xiao-Ping Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Abstract: Febrile seizure (FS) is common in childhood and can impair cognitive function. The potential to exhibit plasticity at many synapses appears to be modulated by prior synaptic activity. This intriguing higher-order form of plasticity has been termed metaplasticity. Plasticity and metaplasticity have been considered to be one of the most important neurological fundaments of learning and memory. In the present study, field potential recording was carried out to detect the effects of FS on plasticity and metaplasticity in the lateral perforant path of rat hippocampus. Brain slices from rat pups of FS model were prepared and superfused. The recording electrodes were placed within the outer molecular layer for recording of lateral perforant path field excitatory postsynaptic potentials (fEPSP). Stimulation of the lateral perforant path and the dentate hilar region was carried out by placing bipolar stimulating electrodes within the outer molecular layer and hilus, respectively. The results showed that long term potentiation (LTP) of control and FS rats didn’t show significant difference after 100 Hz conditioning stimulation. Subjected to 10 Hz priming stimulation applied to lateral perforant path or dentate hilar region 40 min prior to 100 Hz conditioning, the LTP of control group was inhibited, while the LTP of FS rats remained constant. Normalized fEPSP slope 1 h after tetanization of control group was 1.10 ± 0.26 and 1.15 ± 0.14 after homosynaptic and antidromic priming stimulation respectively. On the contrast, FS group didn’t show any depression of LTP after homosynaptic and antidromic priming stimulation, normalized fEPSP slope 1 h after tetanization being 1.35 ± 0.2 and 1.47 ± 0.19, respectively. These results suggest that FS would impair lateral perforant path metaplasticity without affecting LTP. These findings represent an intriguing phenomenon of FS-caused brain damage and imply the injury of excitatory status in different pathways.

Key words: febrile seizure, hippocampus, synaptic plasticity, priming stimulation

热性癫痫发作大鼠海马齿状回外侧支的可塑性和再可塑性

张炼*，罗小平华中科技大学同济医学院附属同济医院儿科，武汉 430030

摘要：热性癫痫发作是儿童常见病，能损害认知功能，而突触可塑性和再可塑性(metaplasticity)是维系大脑认知功能的重要神经基础。本文通过脑片灌流和细胞外场电位记录术研究了热性癫痫发作大鼠海马齿状回外侧支的突触可塑性和再可塑性。制作对照组和热性癫痫发作组大鼠的脑切片后，记录电极置于齿状回外侧支的外分子层获取兴奋性突触后电位。双极刺激电极根据不同需要分别安放于齿状回外侧支或门区。结果显示，大鼠海马齿状回外侧支给予100 Hz的条件刺激后，对照组和热性癫痫发作大鼠海马齿状回外侧支长时程增强(long-term potentiation, LTP)的幅度均没有显著改变。而在齿状回外侧支给予10 Hz的预先刺激，再施加相同的条件刺激后，对照组外侧支LTP幅度明显下降，条件刺激后1 h的相对兴奋性突触后电位(field excitatory postsynaptic potentials, fEPSP)为1.10 ± 0.26；而热性癫痫发作组则表现出明显的LTP，条件刺激后1 h的相对fEPSP为1.35 ± 0.2，与对照组比较有显著性增强(P < 0.05)。在齿状回门区实施10 Hz的预先逆行性刺激，再施加与前述实验相同的条件刺激后，对照组外侧支LTP幅度亦出现明显下降，条件刺激后1 h的相对fEPSP为1.15 ± 0.14；而热性癫痫发作组表现出明显的LTP，条件刺激后1 h的相对fEPSP分别为1.47 ± 0.19，与对照组比较有显著性增强(P < 0.05)。以上结果表

Received 2010-11-09 Accepted 2011-01-05 This work was supported by the National Natural Science Foundation of China (No. 30872796). *Corresponding author. Tel: +86-20-38076224; E-mail: [email protected] ZHANG Lian et al.: Plasticity and Metaplasticity of LPP in Hippocampal DG in Rat Model of FS 125

明，热性癫痫发作可以在不改变突触可塑性的情况下影响再可塑性，从而导致神经损伤，该现象提示热性癫痫发作能通过不同途径增加神经兴奋性。

关键词：热性癫痫发作；海马；突触可塑性；预先刺激中图分类号：Q42

Febrile seizure (FS) is the most common disease in On postnatal (P) days 10 to 12, a group of three male young childhood, affecting 2% to 4% of children be- Sprague-Dawley (SD) rat pups were placed into a fore age 5 years. One third of those with FS have recur- chamber in which hyperthermia was induced by a regu- rent FS by age 6 years, and the incidence of subsequent lated stream of heated air from an adjustable hair dryer epilepsy is 2% to 8%[1]. The causal relationship be- located 50 cm above the container[6]. All rats were pur- tween FS and subsequent epilepsy and cognitive defi- chased from experimental animal center of Tongji cits has always been a focus of intense investigation. Medical College of Huazhong University of Science Hippocampus was believed to be a key point in the and Technology. Meanwhile, the chamber was main- mechanism of FS-induced brain damage. The patho- tained at 37 ºC with water bath. The rectal temperature logical change of hippocampus would affect the cogni- of the rat pup was measured at baseline, every 2 min tive function and learning ability of children. Dentate during hyperthermia, and at the onset of behavioral sei- gyrus is the main part of hippocampus. It is an impor- zures. When rectal temperatures reached approximately tant storage area of mammalian endogenous neural 40 ºC to 41 ºC, the pups became agitated and showed stem cell. Early-life FS causes a dimorphic cytogenic head bobbing or shaking, clonic twitching of hindlimbs, response that results in an increased population of new- behavioral arrest or biting, and chewing of limbs (stage born dentate gyrus cells in young adult male rats[2]. I FS). At 41 ºC to 43 ºC rectal temperature, the pups The elementary feature of most CNS synapses is the exhibited generalized myoclonic jerk, wide-running, ability to modify synaptic strength in an activity-depen- ataxic walking, circling, falling, or tonic-clonic seizures dent way, either as long-term depression (LTD) or long- of limb (stage II FS). After 10 min in the chamber at 40 ºC term potentiation (LTP). The properties of different to 43 ºC rectal temperature, the pups were removed im- forms of LTP and LTD in the rodent hippocampus, and mediately and placed on a cool surface until they re- their molecular underpinnings, have been exceedingly gained posture and their core temperature returned to well studied[3]. A less well studied but particularly in- baseline. The rat pups were divided into two groups: (1) triguing finding is that the capacity of many synapses one FS daily from P10 to P12 (three FS total); and (2) for plastic changes itself is subjected to considerable the same number of rats in control group. The controls activity-dependent variation, or plasticity. This higher- were removed from the cage together with FS group order plasticity has been termed metaplasticity[4]. Inter- during the experiment, but were not subjected to the estingly, induction of metaplasticity does not necessari- hyperthemia. Pups on P10 to P12 per dam were used ly lead to changes in synaptic strength per se, but rather and housed with a 12-h light/dark schedule. The pups modifies the ability of synapses to exhibit synaptic were weaned on P21 and housed in groups. This study plasticity in response to subsequent episodes of synap- was approved by the Animal Care Committee at Hua- tic activity. To keep synapses within a dynamic func- zhong University of Science and Technology. tional range and therefore to prevent them from enter- 1.2 Slice preparation ing states of saturated LTP or LTD, metaplasticity acts P30 male SD rats were anesthetized with diethylether as a crucial factor in normal neuronal function[5]. and decapitated. The brain was removed and immedi- Here, we investigated plasticity and metaplasticity in ately immersed in ice-cold oxygenated (95% O2/5% lateral perforant path of dentate gyrus in a rat model of CO2) dissection solution which contained (in mmol/L): FS. We further demonstrated how early life FS impaird NaCl 125.0, KCl 3.0, CaCl2 0.2, MgSO4 5.0, NaH2PO4 plasticity and metaplasticity in that pathway. 1.25, NaHCO3 26.0, and D-glucose 13.0, pH 7.35. Transverse slices (400 µm) were prepared with a vi- 1 MATERIALS AND METHODS bratome (VT1000S; Leica, Wetzlar, Germany) and transferred to an interface chamber where they were 1.1 Experimental FS in rat pups continuously superfused (1.8 mL/min) with artificial 126 Acta Physiologica Sinica, April 25, 2011, 63(2): 124–130

CSF (ACSF) containing (in mmol/L): NaCl 125.0, KCl conditioning stimulation was carried out with the same

3.0, CaCl2 2.5, MgCl2 1.3, NaH2PO4 1.25, NaHCO3 number of stimuli and spacing of episodes at frequen- 26.0, D-glucose 13.0, pH 7.35, osmolality 300–308 cies of 100 Hz. Both priming and conditioning stimula- mOsm/kg. The temperature of the recording chamber tion were carried out with a stimulation strength in- was maintained at 35 ºC. The slices were allowed to re- creased twofold compared to the baseline stimulation cover for at least one hour before the start of recording. strength. In those cases in which priming stimulation 1.3 Electrophysiology caused a significant change of fEPSP amplitude, the Extracellular borosilicate glass recording microelec- stimulation strength was adjusted to pre-priming baseline levels during the last 20 min before applying a trodes (~2 MΩ) were filled with extracellular ACSF. conditioning tetanus. The anatomic structure and signal The recording electrodes were placed within the outer pathways of hippocampus, the electrodes configuration molecular layer of lateral perforant path to record field and the characters of all three kinds of stimulation were excitatory postsynaptic potentials (fEPSPs). The posi- summarized in Fig. 1. tions of the electrodes were subtly adjusted until lateral perforant path responses showed paired-pulse facilita- 1.4 Drugs and reagents tion (40 ms interpulse interval). Stimulation was car- All salts were obtained from Sigma (Deisenhofen, Ger- ried out with square impulses of 50–200 µA lasting 0.1 many). They were dissolved in double-distilled water ms via a stimulus isolator (WPI, Sarasota). Data were as a stock solution and then diluted in ACSF to a final digitized at 10 kHz and analyzed with pCLAMP 8 soft- concentration before use. ware (Axon Instruments). Stimulus response curves 1.5 Data analysis were performed at the beginning of each experiment. To analyze synaptic efficacy, the maximal slope was The stimulus strength was then adjusted to yield determined in the rising phase of fEPSPs. Values from fEPSPs that were 40%–60% of the maximum response, three successive fEPSPs were averaged to yield one and stimuli were applied at a frequency of 0.025 Hz for data point. To determine the amount of LTP, we first the remainder of the experiment. In some experiments, determined the average value of the fEPSP slope of the antidromic stimulation was carried out by placing a three last fEPSPs during the follow-up period, i.e., 60 stimulating electrode within the dentate hilar region. min after conditioning stimulation. All values were Similar to our previous study[7], priming stimulation normalized to the average fEPSP slope of the first 10 in vitro was carried out at 10 Hz (10 episodes of 20 min of the baseline recording period. All data are ex- pulses, spaced 1 s apart) in all experiments. Subsequent pressed as means ± SEM. Statistical comparisons were

Fig. 1. Schematic diagram showing the structure and signal pathways of hippocampus, the characters of stimulation and the configuration of homosynaptic and antidromic priming stimulations. The arrows in the left part indicate the direction of signal pathways in hippocampus. LPP represents lateral perforant path, and MPP represents media perforant path of dentate gyrus. R and S1 represent recording electrode and stimulating electrode which placed at LPP respectively. S2 represents stimulating electrode which performs antidromic priming stimulation to the axons of granule cells. The right part summarized three kinds of stimulation paradigms applied at S1. ZHANG Lian et al.: Plasticity and Metaplasticity of LPP in Hippocampal DG in Rat Model of FS 127 carried out using Mann-Whitney U test with P < 0.05 ing the same 100 Hz conditioning stimulations as in as the threshold for statistical significance. Fig. 2A. Clearly, priming stimulation caused a dramatic loss of the ability of lateral perforant path synapses to 2 RESULTS undergo potentiation in control group. As shown in Fig. 3A, conditioning stimulations at 100 Hz did not induce 2.1 FS do not alter synaptic plasticity in the lateral LTP in control group (normalized fEPSP slope at 1 h af- perforant path ter tetanization, 1.10 ± 0.26, n = 9). On the other hand, We have initially examined the amount of synaptic the LTP induction of FS group was not affected by 10 plasticity elicited by 100 Hz conditioning stimulation Hz priming stimulation (normalized fEPSP slope at 1 h (10 trains of 20 pulses spaced 1 s apart, frequencies of after tetanization, 1.35 ± 0.21, n = 9). Statistical differ- pulses within trains of 100 Hz). As shown in Fig. 2A, ence was found between the two groups and the result LTP of both control and FS groups were induced by was summarized in Fig. 3B. The experiment indicated 100 Hz stimuli and lasted 1 h. Normalized fEPSP that FS rats lost the ability to exhibit metaplasticity. slopes of FS and control groups at 1 h after tetanization 2.3 Antidromic priming stimulation inhibits subse- were 1.39 ± 0.13 (n = 9) and 1.48 ± 0.12 (n = 8) respec- quent LTP of control but not FS group in the lateral tively. The potentiation of control group was slightly perforant path higher, but no significant difference with FS group was Finally, we examined whether synaptic activation is found. The results were summarized in Fig. 2B. necessary for establishing the priming effect, or wheth- 2.2 Homosynaptic priming stimulation inhibits sub- er inducing postsynaptic neurons to fire at 10 Hz is also sequent LTP of control but not FS group in the lat- sufficient. We placed a bipolar electrode within the eral perforant path dentate hilar region in order to elicit antidromic action We next addressed the question whether priming stimu- potentials in granule cells (Fig. 1). To exclude the pos- lation of the two groups alterd synaptic plasticity in the sibility that the antidromic stimulation might recruit lateral perforant path. We tried to apply the priming progressively less granule neurons during the course of stimulation at a frequency of 10 Hz. We chose this pro- 10 Hz stimulation, we measured the amplitude of the tocol because it did not cause significant long-term antidromic population spike during priming stimula- changes in synaptic transmission. After establishing a tion. The amplitudes of the antidromic population stable baseline, a 10 Hz priming stimulation was ap- spikes elicited by the 1st and 20th stimulus during plied (indicated by empty arrow in Fig. 3A). At 40 min priming stimulation, however, did not reveal a reduc- after priming stimulation, we tested the potential of the tion in amplitude (20th population spike was 97.42% of lateral perforant path to measure synaptic plasticity us- 1st population spike, n = 9, data not shown). Antidro-

Fig. 2. Febrile seizure do not alter synaptic plasticity in the lateral perforant path. A: Time course of the fEPSP slope measured in the lateral perforant path. A 100 Hz tetanus was used as a conditioning stimulus at 0 min. Inset represents sample traces obtained at the different time points indicated by “a” and “b”. B: Summary of the amount of synaptic plasticity measured at 60 min after application of the conditioning tetanus (NS indicates no significant difference). 128 Acta Physiologica Sinica, April 25, 2011, 63(2): 124–130 mic priming stimulation—as homosynaptic priming 3 DISCUSSION one—also resulted in a potent inhibition of subsequent LTP in control group (normalized fEPSP slope at 1 h FS is the most common seizure disorder in childhood, after tetanization, 1.15 ± 0.14, n = 9) compared to FS but its long-term effects on the developing brains, espe- group recordings (normalized fEPSP slope 1 h after tet- cially neuronal injury and neurocognitive function, re- anization, 1.47 ± 0.19, n = 8) (P < 0.05, Fig. 4). This main unresolved. A small fraction of children with FS experiment suggests that FS changed the metaplasticity appear to develop cognitive impairments. Prolonged FS in the lateral perforant path. in early life has long-lasting effects on the hippocampus and can induce cognitive deficits[8].

Fig. 3. Homosynaptic priming stimulation inhibits subsequent LTP of control but not FS group in the lateral perforant path. A: Time course of the fEPSP slope measured at lateral perforant pathway. At −40 min a 10 Hz priming was applied to LPP (Indicated by empty arrow). A 100 Hz tetanus to the lateral perforant path was used as a conditioning stimulus at 0 min. Inset represents sample traces obtained at the different time points indicated by “a” and “b”. B: Summary of the amount of synaptic plasticity measured at 60 min after application of the conditioning tetanus (Asterisk indicates P < 0.05).

Fig. 4. Antidromic priming stimulation inhibits subsequent LTP of control but not FS group in the lateral perforant path. A: Time course of the fEPSP slope measured at the lateral perforant pathway. At −40 min a 10 Hz priming stimulation was applied to dentate hilar region (Indicated by empty arrow). A 100 Hz tetanus to the lateral perforant path was used as a conditioning stimulus at 0 min. Inset represents sample traces obtained at the time points indicated by the lowercase letters (a, b) in the panel. B: Summary of the amount of synaptic plasticity measured at 60 min after application of the conditioning tetanus (Asterisk indicates P < 0.05). ZHANG Lian et al.: Plasticity and Metaplasticity of LPP in Hippocampal DG in Rat Model of FS 129

A recent study has revealed that enhanced CA1 LTP ments should be carried out to investigate the possible and mild mossy fiber sprouting occur after experimen- relative cognitive impairment using different levels of tal FS, without affecting spatial learning and memory FS animal models. in the Morris water maze[9]. These alterations implied Despite a lot of electrophysiological and behavioral that the impaired plasticity didn’t always couple with evidences indicated the FS caused brain damage[8], the the deficits of cognitive functions. Probably the brain pathological changes didn’t occur at the same severity damage was related to the degree of repetitive FS, as layer. Notenboom et al.[9] reported that enhanced CA1 described by Chang et al.[10]. Another complicated fac- LTP didn’t affect spatial learning and memory in the tor involved is the maturity of the developing dentate Morris water maze in a rat model of FS. Recurrent ear- gyrus neurons. Ye et al.[11] reported the different elec- ly-life seizures might differ from single seizures in their trophysiological character of developing dentate gyrus effects on long-term cognitive function. Critical param- granule cells. We therefore hypothesized that FS- eters included age, the number of seizures, their severi- caused neurotoxicity may affect individual neuronal ty and distribution[13]. The impairment of cognitive functions in distinctive ways and with variable degrees function and plasticity were significantly detectable. of reversibility. Here we applied a relative mild stimulation regime A nonlinear relationship between post-synaptic activ- compared to that in Chang et al.[6] to establish animal ity and the direction and amount of synaptic long-term models. We found the FS-related traces were too small modifications was postulated by Bienenstock, Cooper to observe by routine LTP examination at the present and Munro[12]. The threshold level at which neither LTP condition and the results helped us to reveal a deeper nor LTD was elicited, generally termed θm, was found layer of the disease. to be close to 10 Hz in our former experiments[7]. This A few researches concerning about the relationship LTD/LTP response function was dramatically altered between FS and plasticity were carried out and revealed following application of a priming stimulation in the 10 multiple factors were involved. Dube[14] reported that Hz range (close to θm), which does not itself signifi- neuropeptide Y expression was up-regulated in hip- cantly alter synaptic strength. In the classical view, the pocampus after experimentally induced FS, and neuro- expression mechanism of metaplasticity underlying peptide Y up-regulation was associated with an in- these phenomena was hypothesized to be a shift in θm, creased seizure threshold for additional (recurrent) FS, with the maximal amount of depression or potentiation and this effect was abolished when an antagonist left intact. Our previous study suggested that metaplas- against neuropeptide Y receptor type 2 was applied. ticity may also be expressed as a change in the maxi- These findings suggest that inhibitory actions of neuro- mal amount of LTD or LTP synapses can exhibit[7]. peptide Y, released after seizures, exert a protective ef- In the present study, we examine three specific mea- fect that reduces the risk of seizure recurrence in the sures of hippocampal lateral perforant path plasticity/ developing brain. Another experiment[15] of endocan- metaplasticity in rats with a prior history of FS: (i) ac- nabinoid signaling at perisomatic GABAergic synapses tivity-dependent synaptic plasticity (LTP); (ii) homos- demonstrated that blocking the induction of this plas- ynaptic metaplasticity; and (iii) antidromic metaplastic- ticity abolishes the long-term effects of prolonged FS ity in the lateral perforant path of hippocampal dentate in the developing brain. Furthermore, some evidences gyrus. Our experiment demonstrated that FS didn’t im- suggested that extracellular matrix components regulat- pact LTP in lateral perforant path with the present stim- ing physiological plasticity/metaplasticity were also ulation paradigms. Intriguingly, LTP of control groups engaged in different aspects of epileptogenesis[16]. were significantly depressed by a mild 10 Hz homosyn- In summary, we report that FS cause the loss of in- aptic or antidromic priming stimulation, while LTP of hibitory effect in the lateral perforant path after 10 Hz FS animals remained unaffected. The results showed priming stimulation, i.e. attenuate metaplasticity. This that FS could cause a subtle change to synaptic meta- form of electrophysiological change is subtle because plasticity without affecting plasticity. The final outcome synaptic plasticity is not affected. Furthermore, the loss of the synapses would be the imperceptible increase of of metaplasticity appears with both homosynaptic and neuro-excitability. The functional aspects of the meta- antidromic priming stimulation. These findings repre- plasticity loss remain unclear. 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