- Email: [email protected]
Opposite effects of lead exposure on taurine- and HFS-induced LTP in rat hippocampus
Brain Research Bulletin 64 (2005) 525–531
Opposite effects of lead exposure on taurine- and HFS-induced LTP in rat hippocampus Kuai Yu, Shan-Shan Yu, Di-Yun Ruan∗ School of Life Science, University of Science and Technology of China, Hefei, Anhui 230027, PR China Received 20 December 2003; received in revised form 12 May 2004; accepted 9 November 2004 Available online 7 December 2004
Abstract The effect of lead exposure on taurine-induced long-term potentiation (LTPTAU ) was examined and compared with high-frequency stimulation-induced one (LTPHFS ). Field excitatory postsynaptic potentials (fEPSP) and fiber volley (FV) in area CA1 of hippocampal slice were recorded in control and lead-exposed rats. In contrast to the inhibitory effects of lead exposure on LTPHFS , the amplitude of LTPTAU in the lead-exposed rats (199.3 ± 13.7%, n = 12) was significantly larger than that in controls (152.3 ± 17.0%, n = 12). It was also observed that taurine induced greater FV potentiation in lead-exposed rats (162.6 ± 9.0%, n = 10) than controls (132.1 ± 6.9%, n = 11). In addition, after a previous HFS, sequent perfusion of taurine could further increase the synaptic efficacy in lead-exposed rats. These results provide the first evidence that chronic lead exposure has opposite effects on the two types of LTP resulting from different lead toxicity sites. © 2004 Elsevier Inc. All rights reserved. Keywords: Lead; Taurine; Long-term potentiation; Hippocampus; Synaptic plasticity
1. Introduction In mammalian brain, taurine is one of the most abundant free amino acids [8]. In central neural system, its physiological actions are considered essential in development [20], osmoregulation [15] and neurotransmission [14]. Recently, Galarreta et al. [5] have reported that taurine application generates a long-term potentiation (LTP) of excitatory synaptic potentials in the hippocampus, which is independent of GABAA and NMDA receptor activation. On the basis of its novel capability, taurine could be proposed as a retrograde factor involved in LTP maintenance [16]. Persistent changes in the hippocampal synaptic strength seem to be especially relevant phenomena accounting for the cellular basis of learning and memory [11]. Although the previous study [16] has shown that LTPHFS and LTPTAU occlude ∗
Corresponding author. Tel.: +86 551 3606374; fax: +86 551 3601443. E-mail addresses: [email protected], [email protected] (D.-Y. Ruan). 0361-9230/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2004.11.005
mutually and therefore may share some mechanisms involved in LTP, LTPTAU has some unusual properties, such as the lack of pathway specificity, the dependence on low-voltage activated calcium channels (LVACC) and taurine uptake system. Moreover, unlike LTPHFS , LTPTAU is a not only postsynaptic but also presynaptic phenomenon. As a well-known environmental toxicant, lead has been concerned for its severe impairment on the cognitive function in children [3]. A great deal of studies reported that chronic developmental lead exposure raises the threshold for and impairs the induction of LTP of excitatory postsynaptic potentials (EPSPs) in rat hippocampus [6,10,17]. And, it has also been suggested that the inhibition of LTPHFS is due to the NMDA receptor and voltage-sensitive calcium channels being blocked by lead [1,2]. Since LTPTAU differs from LTPHFS in the dependence on NMDA receptor and calcium channels, we wonder whether lead exposure has different effects on LTPTAU . In order to provide evidence of the hypothesis, we examined the effects of chronic lead exposure on LTPTAU and compared them with those on LTPHFS .
526
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
2. Materials and methods 2.1. Experimental animals The protocol for chronic lead exposure to lead has been described previously [17]. Briefly, on parturition day, Wistar dams were randomly divided into two groups: control and lead-exposed. The lead-exposed pups acquired lead via milk of dams whose drinking water contained 0.2% (1090 ppm) lead acetate from parturition to weaning, while the control dams remained on distilled water throughout the lactation period. At age of 21 days, offspring were weaned, housed in a colony room with a 12:12 light:dark schedule and permitted free access to food and distilled water. For any given experimental measure, equal numbers of females and males were used and no more than two pups were sampled from the same litter. 2.2. Blood and hippocampal lead determinations Blood and hippocampal lead determinations were made in littermates of the animals utilized for electrophysiology on the day that recordings were made. Blood samples (2 ml per animal) were collected in heparinized syringes via cardiac puncture of anesthetized rats. After decapitation, two hippocampi were collected. Lead concentrations were measured by a plasmaQuad3 plasma mass spectrograph (VG Elemental, UK) after the tissues were digested with an organic tissue solubilizer. 2.3. Slice preparation and drugs Guidelines contained in NIH publication 80–23 revised in 1978 on the principles of laboratory animal care were followed throughout. Experiments were carried out on rats at the age of postnatal 25–30 days (weight: 40–50 g). After the rat was decapitated, its brain was rapidly removed and dropped into ice-cold, oxygenated (95% O2 /5% CO2 ), artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 124, KCl 5, NaH2 PO4 1.25, NaHCO3 26, MgCl2 1.5, CaCl2 2.5, d-glucose 10, pH 7.30–7.45. Then, the hippocampi were dissected from the surrounding tissue, and 400-m-thick transversal slices were cut by a manual chopper. Before recording, the slices were placed in an interface holding chamber where they were maintained in oxygenated ACSF at room temperature (21–25 ◦ C). After at least 1 h, a slice was transferred to a submersion-type recording chamber (BSCHT Medical Systems, USA), in which it was superfused continuously with 30–32 ◦ C oxygenated ACSF at the rate of 1 ml/min. Drugs applied in addition to the standard ACSF included taurine and picrotoxin. All the drugs used were from Sigma.
pulses (0.1–0.6 mA, 20 s, 0.05 Hz), applied through bipolar microelectrodes located in stratum radiatum. Evoked extracellular field potentials (fEPSP and FV) from the stratum radiatum of the CA1 region were recorded with low resistance glass micropipettes filled with 2 M NaCl (1–2 M), that were connected to field effect transistors, the outputs of which were filtered between 1 and 3000 Hz. In those experiments in which the GABAA antagonist picrotoxin (100 M) was present in the bath medium, a cut was made between CA1 and CA3, and the concentrations of Ca2+ and Mg2+ were increased to 4 mM to prevent epileptiform discharges. Consistent with the previous report [5], these conditions (surgical cut and high concentrations of Ca2+ and Mg2+ ) did not affect taurine effects on fEPSP, which was observed in our prior experiments. 2.5. Data analysis All statistical analyses were performed using the EXCEL package and Origion 7.0 in personal computers. The amplitude of the presynaptic FV was measured from the baseline to the negative peak of the FV. The initial rising slope of fEPSP was extracted as the measure amplitude of a response. Data were normalized with respect to the mean values of the responses at the 20 min control period (in standard ACSF), before the application of taurine or HFS. Results were expressed as mean ± S.E.M. Statistical differences were assessed by one- or two-way analyses of variance (ANOVA), and two-tailed Student’s t-test for multiple mean comparisons. Differences were considered significant at P ≤ 0.05. In the representation, n was the number of the animals that were sampled.
3. Results In order to rule out the factor of sex, we previously measured blood and hippocampal lead levels, LTPHFS and LTPTAU in males (n = 8) and females (n = 8), respectively. No observable differences were found in control or lead-exposed rats. 3.1. Blood and hippocampal lead levels Lead concentrations of blood and hippocampus were 7.52 ± 0.43 g/dl and 143.4 ± 19.3 ng/g in 16 control rats and 33.0 ± 0.23 g/dl and 364.4 ± 28.5 ng/g in 16 lead-exposed rats. Lead levels of blood and hippocampus in the leadexposed rats were significantly higher than those in controls (n = 16, P < 0.001).
2.4. Recording
3.2. Opposite effects of chronic lead exposure on the induction of LTPHFS and LTPTAU
To obtain evoked synaptic responses in CA1 area, Schaffer collateral-commissural fibers were stimulated with electrical
EPSPs were recorded for more than 65 min after HFS or taurine application. In the subjects used for LTP induction,
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
527
much greater potentiation of EPSPs developing in this overlapped duration. 3.3. Lead exposure enhances both FV and synaptic efficacy potentiations induced by taurine As shown in Fig. 2A(b and c), taurine-induced potentiations consisted of the increases of both FV amplitude and synaptic efficacy, which was different with LTPHFS (Fig. 2A(a)). Therefore, we examined the effect of lead exposure on these two factors. In some experiments, we were able to record the FV, which is a consequence of the action potentials generated by the electrical stimulation of the Schaffer collaterals axons. Fig. 2B showed that the FV amplitude had a significantly (P < 0.001) higher increasing level in lead-exposed rats (162.6 ± 8.9%, n = 10) than in controls (131.9 ± 6.9%, n = 11) at 1 h after taurine withdrawal. In another group of experiments (Fig. 2C), after 20 min of taurine withdrawal, the stimulus strength was reduced until FV amplitude matched the pre-taurine amplitude. It was observed that the fEPSP slope still remained greater in lead-exposed rats (144.5 ± 10.0%, n = 10) than in controls (124.9 ± 10.7%, n = 11). 3.4. Taurine induces the further potentiation after a previous saturated LTPHFS merely in lead-exposed subjects
Fig. 1. Lead exposure has opposite effects on the inductions of LTPHFS (A) and LTPTAU (B). HFS (100 Hz, 1 s) and taurine (10 mM, 30 min) were both applied on the slices of 12 control and 12 lead-exposed subjects. The recovery and enhancement of EPSP slopes were recorded at least 65 min after HFS and taurine application. For the sake of clarity, points are plotted every 2 min. Here and in subsequent figures up-arrow bar (↑) indicates the application of HFS and horizontal bar ( ) indicates the period of taurine perfusion. The inserted histograms represent the amplitudes of LTP as the mean increase in EPSP slope at 50–60 min in relation to baseline response after HFS and taurine application in the control and lead-exposed subjects (summary of the results in A and B). Asterisks indicate significant differences (P < 0.01) compared with control values.
lead exposure had no significant effect on the original potential trace during baseline measures. Fig. 1A and B (HFS and taurine, respectively) illustrate the responses to LTP induction in the 12 control and 12 lead-exposed rats. It is clear that while lead attenuates the induction of LTPHFS , it enhances the induction of LTPTAU . And, as summarized in the insert of Fig. 1A, LTPHFS amplitudes were significantly (P < 0.001) lower in lead-exposed rats (121.1 ± 8.9%) compared to controls (180.9 ± 15.9%). However, LTPTAU amplitudes were significantly higher in lead-exposed rats (199.3 ± 13.7% versus 152.3 ± 17.0%, P < 0.001). It should be noted that at the beginning of 10 mM taurine perfusion, a balance between the inhibitory action of taurine on GABAA receptors and the potentiation effect could exist. Therefore, the diminished effect of taurine on initial response suppression in the lead-exposed animal could be a result of a
del Olmo et al. [16] have demonstrated that in those synapses from which prior HFS-LTP has developed, taurine does not induce long-lasting increase in synaptic efficacy. However, the observation on lead-exposed subjects was not as such. Since GABAA receptors are not involved in taurine-induced potentiation [5], we performed a group of experiments with the presence of picrotoxin to facilitate and maximize LTP induction. A saturated LTP was induced by two 100 Hz, 1 s trains (spaced 120 s). Once the potentiation stabilized (30 min after the trains), the stimulus strength was reduced until a fEPSP of the similar pre-tetanus size was obtained. Then, after a new 10 min baseline period, 10 mM taurine was bath perfused for 30 min. When this maneuver was performed (Fig. 3A), it was observed that the fEPSP slope measured 25–29 min after trains was smaller in lead-exposed rats (128.2 ± 11.2%, n = 9) than in controls (175.5 ± 8.7%, n = 10), but following taurine withdrawal, it increased greater in lead-exposed rats (166.0 ± 11.8%) than in controls (123.1 ± 11.0%). In another set of experiments (see Fig. 3A), field potentials were evoked by increasing stimulus strength in 10 A steps. They were recorded at times as follows: before LTPHFS induction, 30 min thereafter, and 50 min after taurine withdrawal. Both control (n = 6) and lead-exposed subjects (n = 6) were examined. For each slice, fEPSP versus FV plots in the different recording duration were depicted. The representative fEPSP/FV plots from a control and a lead-treated slice were shown in Fig. 3B. It was illustrated obviously that the increase in synaptic efficacy (a
528
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
Fig. 2. The influences of lead exposure on both pre- and post-synaptic potentiation induced by taurine. (A) The traces in a represented fEPSP recorded in a slice from control animal before and after the inducement of LTPHFS during one of the experiments used for Fig. 1A. Traces in b and c were recorded in a control slice at different times (baseline, b and c) during one of the experiments used for graph C. (B) The taurine-induced increases of FV amplitude were significantly greater in lead-exposed subjects (n = 10) than in controls (n = 11). (C) To exclude the effects of FV, stimulus strength was reduced (indicated by an arrow) to size-match the FV amplitude with the baseline values after 20 min of taurine withdrawal. The fEPSP slope still remained greater in lead-exposed rats (n = 10) than in controls (n = 11).
leftward shift in the curve), which was induced by HFS, was not further changed in the controls after the taurine perfusion, while a clear increase in synaptic efficacy was evoked by taurine in the lead-exposed rats.
4. Discussion According to the previous report [5], we have known that taurine-induced synaptic potentiation consists of two long-
lasting factors: (1) the increase in the synaptic efficacy detected by a shift to the left in the fEPSP/FV curves; and (2) the enhancement of axon excitability detected by the increase of FV amplitude. The principle finding reported in this paper is that the two factors are both enhanced in the lead-exposed animals. And, it is very different from the inhibitory effect of lead-exposure on LTPHFS in which the latter factor is not involved. Since both LTPTAU components have similar extraand intra-cellular Ca2+ requirements for their maintenance
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
529
Fig. 3. After a previous LTPHFS , the subsequent taurine perfusion evoked the further potentiation of synaptic efficacy merely in the lead-exposed subjects. (A) The upper traces showed representative fEPSPs recorded from a control slice at different times (a, b, c and d on the graph) during one of the experiments used for the graph. For comparison, trace a and c were superposed, respectively, with b and c, and d. The graph plot represented the temporal evolution of fEPSP slope changes recorded from control and lead-exposed subjects. After two tetani (100 Hz, 1 s) at 2 min intervals was applied to induce a saturated LTP, at the upside down arrowhead the stimulus strength was reduced until a fEPSP of similar pre-tetanus size was obtained. Then, taurine (10 mM) was bath perfused during the time indicated by the black horizontal bar. The upside up arrowhead indicates the moment when the stimulus strength was readjusted the same as in the baseline period. (B) Representative plots of fEPSP slope vs. FV amplitude evoked in a control and a lead-exposed subject, respectively. They were recorded at times as follow: before LTP induction (䊉), 30 min thereafter () and 50 min after taurine withdrawal (). Data correspond to two experiments performed as those summarized in A but were not included in that graph.
mechanisms [16], the most probable explanation for our finding is deduced from the effects of lead on calmodulin and protein kinases. The dependence on the rise of [Ca2+ ]i is a common property for LTPTAU [16] and other types of long-lasting synaptic potentiation. And, several families of Ca2+ -sensitive kinases are involved in hippocampal LTP [12]. One mechanism by which Pb2+ appears to alert cellular physiology is replacing Ca2+ in Ca2+ -binding proteins [4], where Pb2+ may acts as an agonist, partial agonist or antagonist of the normal action of Ca2+ . For example, Pb2+ has been reported to be a fully effective agonist in activating
calmodulin [7]. However, in activating conventional Ca2+ dependent protein kinase C (PKC), Pb2+ appears to be a partial agonist, stimulating PKC at low concentration (less than about 10 nM), but to a lesser extent than Ca2+ does, inhibiting PKC at high concentrations (greater than about 100 nM), and possibly reducing the stimulatory effects of high free Ca2+ concentrations [21]. The varied effects may result from the multiple Ca2+ -binding sites of Ca2+ -activited proteins, all of which must normally be occupied by Ca2+ for full activity. When Pb2+ and Ca2+ coexist around, those sites on a single protein may be occupied solely by Ca2+ ,
530
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
solely by Pb2+ , or possibly by a combination of both metals, depending on their absolute and relative concentrations [21]. The additive effects of Pb2+ and Ca2+ may cause important consequences for the protein activity. Some PKC- and calmodulin-stimulated proteins, such as non-NMDA receptors, type I adenylyl cyclase, plasma membrane Ca2+ -ATPase and calcineurin, are partially activated at the typical resting intracellular free Ca2+ ion concentration of 50–100 nM, and they may constantly experience enhanced stimulation by low concentrations of intracellular free Pb2+ in Pb2+ -exposed cells [9]. In our results, lead enhances the induction and maintenance of LTPTAU , while it has opposite effects on that of LTPHFS . This discrepancy may result from the different sources and lasting time in [Ca2+ ]i rise. First, to induce LTPTAU , only a small Ca2+ influx through LVACC is required and an amplification of this Ca2+ influx by Ca2+ released from intracellular stores is involved [16]. Unlike taurine, HFS cannot fully raise [Ca2+ ]i due to the NMDA receptor and voltagesensitive calcium channels being blocked by lead [2,10]. Lead cannot activate calmodulin and protein kinases without the cooperation of enough free Ca2+ [9,21]. Second, the activation of calmodulin and protein kinases may depend on the action time of the agonists. The application of taurine lasts more than a dozen minutes, which effectively activates LVACC and may cause a long-lasting [Ca2+ ]i rise [16], while [Ca2+ ]i rise disappears within seconds after HFS [13]. Third, HFS results in localized “hot spots” of free Ca2+ near the mouths of intracellular release channels or plasma membrane influx channels, in which the free Ca2+ ion concentration may reach dozens of micromolars, with the Ca2+ concentration rapidly decreasing with distance from the site of Ca2+ entry into the cytoplasm. The “hot spots” may differ from the sites of Pb2+ binding. Taurine also causes a rise in [Ca2+ ]i through the intracellular Ca2+ release channels and plasma membrane influx channels. However, the much longer duration of its effect may apply enough time for the dispersion of free Ca2+ throughout a cell, so Pb2+ and Ca2+ can effectively cooperate. Moreover, the presynaptic effect of taurine might be also involved though it is hard to get a clear conclusion by this research. In addition to the maintenance of potentiation, we should also consider the initial short-term potentiation of FV amplitude that was greatly enhanced by lead-exposure. Since intracellular application of taurine can enhance the peak Na+ current [18], Galarreta et al. have suggested a consequence of the decrease in the action potential firing threshold induced by this amino acid [5]. Moreover, chronic lead-exposure could depress the spiking activity in several types of neurons [19]. Therefore, the possibility cannot be ruled out that taurine may directly modulate the Na+ channels and remove the blockage of lead on the fiber excitability. And, a relatively greater FV increase could be expected in lead-exposed subject compared with the control. Alternatively, the lead-induced changes in the neural circuit development might also contribute to our finding.
As discussed in the previous reports [5,16], high content of taurine (5–70 mM) exists in brain cells. Also, taurine movements between extra- and intra-cellular compartments could represent the physiological framework for LTPTAU , which could also be considered as a model for several physiological and pathological situations, such as brain development and osmotic stress. If these assumptions were true, our data would reveal a more extensive interference of lead on neural system. Moreover, if taurine-induced changes in synaptic transmission were also involved in the procedure of learning and memory, our data shown in Fig. 3 would surprisingly suggest a possibility that the reinforcement of taurine might renovate the lead-induced impairment. Nevertheless, based on the present limited understanding about LTPTAU , any consideration about the physiological meaning of our finding would be very speculative. In conclusion, the chronic lead exposure increases both components accounting for LTPTAU (i.e. long-term potentiation in synaptic efficacy and FV amplitude). Moreover, though LTPTAU and LTPHFS utilize some common mechanism for the generation of long-term increases in synaptic efficacy, the quantity of calcium from different sources should be varied and thus induce different contribution to each type of LTP. Therefore, Pb2+ could cause opposite effects on LTPTAU and LTPHFS when cooperating with various [Ca2+ ]i .
Acknowledgements This work was supported by the National Basic Research program of China (No. 2002CB512907), Academic Sinica (No. KZCX3-sw-437), the National Nature Science Foundation of China (No. 30170809, 30300288, 30000039), Research Fund for the Doctoral Program of High Education (RFDP, No. 20020358053), University of Science and Technology of China (No. KB0833, KY1206).
References [1] M. Alkondon, A.C.S. Costa, V. Radhakrishnan, et al., Selective blockade of NMDA-activated channel currents may be implicated in learning deficits caused by lead, FEBS Lett. 261 (1990) 124–130. [2] G. Audesirk, T. Audesirk, Effects of in vitro lead exposure on voltage-sensitive calcium channels differ among cell types in central neurons of Lymnaea stagnalis, Neurotoxicology 10 (1989) 659–669. [3] D. Bellinger, J. Sloman, A. Leviton, et al., Low-level lead exposure and children’s cognitive function in the preschool years, Pediatrics 87 (2) (1991) 219–227. [4] C.S. Fullmer, S. Edelstein, R.H. Wasserman, Lead-binding properties of intestinal calcium-binding proteins, J. Biol. Chem. 260 (1985) 6816–6819. [5] M. Galarreta, J. Bustamante, R. Martin del Rio, et al., Taurine induces a long-lasting increase of synaptic efficacy and axon excitability in the hippocampus, J. Neurosci. 16 (1996) 92–102. [6] M.E. Gilbert, C.M. Mack, S.M. Lasley, Chronic developmental lead exposure increases the threshold for long-term potentiation in rat dentate gyrus in vivo, Brain Res. 736 (1996) 118–124.
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531 [7] E. Habermann, K. Crowell, P. Janicki, Lead and other metals can substitute for Ca2+ in calmodulin, Arch. Toxicol. 54 (1983) 61–70. [8] R.J. Huxtable, Taurine in the central nervous system and the mammalian actions of taurine, Prog. Neurobiol. 32 (1989) 471–533. [9] M. Kern, M. Wisniewski, L. Cabell, et al., Inorganic lead and calcium interact positively in activation of calmodulin, Neurotoxicology 21 (3) (2000) 353–364. [10] S.M. Lasley, M.E. Gilbert, Lead inhibits the rat N-methyl-d-aspartate receptor channel by binding to a site distinct from the zinc allosteric site, Toxicol. Appl. Pharmacol. 159 (1999) 224–233. [11] G. Lynch, M. Baudry, The biochemistry of memory: a new and specific hypothesis, Science 224 (1984) 1057–1063. [12] R.C. Malenka, R.A. Nicoll, NMDA-receptor-dependent synaptic plasticity: multiple forms and mechanisms, Trends Neurosci. 16 (1993) 521–527. [13] R.C. Malenka, R.A. Nicoll, Long-term potentiation—a decade of progress? Science 285 (1999) 1870–1874. [14] W.J. McBride, RC.A. Frederickson, Taurine as a possible inhibitory transmitter in the cerebellum, Fed Proc. 39 (1980) 2701–2705.
531
[15] S.S. Oja, P. Saransaari, Taurine as osmoregulator and neuromodulator in the brain, Metab. Brain Dis. 11 (2) (1996) 153–164. [16] N. del Olmo, M. Galarreta, J. Bustamante, et al., Taurine-induced synaptic potentiation: role of calcium and interaction with LTP, Neuropharmacology 39 (2000) 40–54. [17] D.Y. Ruan, K.F. Yan, S.Y. Ge, et al., Effects of chronic lead exposure on short-term and long-term depression in area CA1 of the rat hippocampus in vivo, Chemosphere 41 (2000) 165–171. [18] H. Satoh, Cardioprotective actions of taurine against intracellular and extracellular calcium-induced effects, Adv. Exp. Med. Biol. 359 (1994) 181–196. [19] B. Scott, J. Lew, Chronic exposure to lead causes persistent alterations in the electric membrane properties of neurons in cell culture, J. Neurobiol. 16 (6) (1985) 425–433. [20] J.A. Sturman, Taurine in development, Physiol. Rev. 73 (1993) 119–147. [21] X. Sun, X. Tian, J.L. Tomsig, et al., Analysis of differential effects of Pb2+ on protein kinase C isozymes, Toxicol. Appl. Pharmacol. 156 (1999) 40–45.
Opposite effects of lead exposure on taurine- and HFS-induced LTP in rat hippocampus Kuai Yu, Shan-Shan Yu, Di-Yun Ruan∗ School of Life Science, University of Science and Technology of China, Hefei, Anhui 230027, PR China Received 20 December 2003; received in revised form 12 May 2004; accepted 9 November 2004 Available online 7 December 2004
Abstract The effect of lead exposure on taurine-induced long-term potentiation (LTPTAU ) was examined and compared with high-frequency stimulation-induced one (LTPHFS ). Field excitatory postsynaptic potentials (fEPSP) and fiber volley (FV) in area CA1 of hippocampal slice were recorded in control and lead-exposed rats. In contrast to the inhibitory effects of lead exposure on LTPHFS , the amplitude of LTPTAU in the lead-exposed rats (199.3 ± 13.7%, n = 12) was significantly larger than that in controls (152.3 ± 17.0%, n = 12). It was also observed that taurine induced greater FV potentiation in lead-exposed rats (162.6 ± 9.0%, n = 10) than controls (132.1 ± 6.9%, n = 11). In addition, after a previous HFS, sequent perfusion of taurine could further increase the synaptic efficacy in lead-exposed rats. These results provide the first evidence that chronic lead exposure has opposite effects on the two types of LTP resulting from different lead toxicity sites. © 2004 Elsevier Inc. All rights reserved. Keywords: Lead; Taurine; Long-term potentiation; Hippocampus; Synaptic plasticity
1. Introduction In mammalian brain, taurine is one of the most abundant free amino acids [8]. In central neural system, its physiological actions are considered essential in development [20], osmoregulation [15] and neurotransmission [14]. Recently, Galarreta et al. [5] have reported that taurine application generates a long-term potentiation (LTP) of excitatory synaptic potentials in the hippocampus, which is independent of GABAA and NMDA receptor activation. On the basis of its novel capability, taurine could be proposed as a retrograde factor involved in LTP maintenance [16]. Persistent changes in the hippocampal synaptic strength seem to be especially relevant phenomena accounting for the cellular basis of learning and memory [11]. Although the previous study [16] has shown that LTPHFS and LTPTAU occlude ∗
Corresponding author. Tel.: +86 551 3606374; fax: +86 551 3601443. E-mail addresses: [email protected], [email protected] (D.-Y. Ruan). 0361-9230/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2004.11.005
mutually and therefore may share some mechanisms involved in LTP, LTPTAU has some unusual properties, such as the lack of pathway specificity, the dependence on low-voltage activated calcium channels (LVACC) and taurine uptake system. Moreover, unlike LTPHFS , LTPTAU is a not only postsynaptic but also presynaptic phenomenon. As a well-known environmental toxicant, lead has been concerned for its severe impairment on the cognitive function in children [3]. A great deal of studies reported that chronic developmental lead exposure raises the threshold for and impairs the induction of LTP of excitatory postsynaptic potentials (EPSPs) in rat hippocampus [6,10,17]. And, it has also been suggested that the inhibition of LTPHFS is due to the NMDA receptor and voltage-sensitive calcium channels being blocked by lead [1,2]. Since LTPTAU differs from LTPHFS in the dependence on NMDA receptor and calcium channels, we wonder whether lead exposure has different effects on LTPTAU . In order to provide evidence of the hypothesis, we examined the effects of chronic lead exposure on LTPTAU and compared them with those on LTPHFS .
526
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
2. Materials and methods 2.1. Experimental animals The protocol for chronic lead exposure to lead has been described previously [17]. Briefly, on parturition day, Wistar dams were randomly divided into two groups: control and lead-exposed. The lead-exposed pups acquired lead via milk of dams whose drinking water contained 0.2% (1090 ppm) lead acetate from parturition to weaning, while the control dams remained on distilled water throughout the lactation period. At age of 21 days, offspring were weaned, housed in a colony room with a 12:12 light:dark schedule and permitted free access to food and distilled water. For any given experimental measure, equal numbers of females and males were used and no more than two pups were sampled from the same litter. 2.2. Blood and hippocampal lead determinations Blood and hippocampal lead determinations were made in littermates of the animals utilized for electrophysiology on the day that recordings were made. Blood samples (2 ml per animal) were collected in heparinized syringes via cardiac puncture of anesthetized rats. After decapitation, two hippocampi were collected. Lead concentrations were measured by a plasmaQuad3 plasma mass spectrograph (VG Elemental, UK) after the tissues were digested with an organic tissue solubilizer. 2.3. Slice preparation and drugs Guidelines contained in NIH publication 80–23 revised in 1978 on the principles of laboratory animal care were followed throughout. Experiments were carried out on rats at the age of postnatal 25–30 days (weight: 40–50 g). After the rat was decapitated, its brain was rapidly removed and dropped into ice-cold, oxygenated (95% O2 /5% CO2 ), artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 124, KCl 5, NaH2 PO4 1.25, NaHCO3 26, MgCl2 1.5, CaCl2 2.5, d-glucose 10, pH 7.30–7.45. Then, the hippocampi were dissected from the surrounding tissue, and 400-m-thick transversal slices were cut by a manual chopper. Before recording, the slices were placed in an interface holding chamber where they were maintained in oxygenated ACSF at room temperature (21–25 ◦ C). After at least 1 h, a slice was transferred to a submersion-type recording chamber (BSCHT Medical Systems, USA), in which it was superfused continuously with 30–32 ◦ C oxygenated ACSF at the rate of 1 ml/min. Drugs applied in addition to the standard ACSF included taurine and picrotoxin. All the drugs used were from Sigma.
pulses (0.1–0.6 mA, 20 s, 0.05 Hz), applied through bipolar microelectrodes located in stratum radiatum. Evoked extracellular field potentials (fEPSP and FV) from the stratum radiatum of the CA1 region were recorded with low resistance glass micropipettes filled with 2 M NaCl (1–2 M), that were connected to field effect transistors, the outputs of which were filtered between 1 and 3000 Hz. In those experiments in which the GABAA antagonist picrotoxin (100 M) was present in the bath medium, a cut was made between CA1 and CA3, and the concentrations of Ca2+ and Mg2+ were increased to 4 mM to prevent epileptiform discharges. Consistent with the previous report [5], these conditions (surgical cut and high concentrations of Ca2+ and Mg2+ ) did not affect taurine effects on fEPSP, which was observed in our prior experiments. 2.5. Data analysis All statistical analyses were performed using the EXCEL package and Origion 7.0 in personal computers. The amplitude of the presynaptic FV was measured from the baseline to the negative peak of the FV. The initial rising slope of fEPSP was extracted as the measure amplitude of a response. Data were normalized with respect to the mean values of the responses at the 20 min control period (in standard ACSF), before the application of taurine or HFS. Results were expressed as mean ± S.E.M. Statistical differences were assessed by one- or two-way analyses of variance (ANOVA), and two-tailed Student’s t-test for multiple mean comparisons. Differences were considered significant at P ≤ 0.05. In the representation, n was the number of the animals that were sampled.
3. Results In order to rule out the factor of sex, we previously measured blood and hippocampal lead levels, LTPHFS and LTPTAU in males (n = 8) and females (n = 8), respectively. No observable differences were found in control or lead-exposed rats. 3.1. Blood and hippocampal lead levels Lead concentrations of blood and hippocampus were 7.52 ± 0.43 g/dl and 143.4 ± 19.3 ng/g in 16 control rats and 33.0 ± 0.23 g/dl and 364.4 ± 28.5 ng/g in 16 lead-exposed rats. Lead levels of blood and hippocampus in the leadexposed rats were significantly higher than those in controls (n = 16, P < 0.001).
2.4. Recording
3.2. Opposite effects of chronic lead exposure on the induction of LTPHFS and LTPTAU
To obtain evoked synaptic responses in CA1 area, Schaffer collateral-commissural fibers were stimulated with electrical
EPSPs were recorded for more than 65 min after HFS or taurine application. In the subjects used for LTP induction,
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
527
much greater potentiation of EPSPs developing in this overlapped duration. 3.3. Lead exposure enhances both FV and synaptic efficacy potentiations induced by taurine As shown in Fig. 2A(b and c), taurine-induced potentiations consisted of the increases of both FV amplitude and synaptic efficacy, which was different with LTPHFS (Fig. 2A(a)). Therefore, we examined the effect of lead exposure on these two factors. In some experiments, we were able to record the FV, which is a consequence of the action potentials generated by the electrical stimulation of the Schaffer collaterals axons. Fig. 2B showed that the FV amplitude had a significantly (P < 0.001) higher increasing level in lead-exposed rats (162.6 ± 8.9%, n = 10) than in controls (131.9 ± 6.9%, n = 11) at 1 h after taurine withdrawal. In another group of experiments (Fig. 2C), after 20 min of taurine withdrawal, the stimulus strength was reduced until FV amplitude matched the pre-taurine amplitude. It was observed that the fEPSP slope still remained greater in lead-exposed rats (144.5 ± 10.0%, n = 10) than in controls (124.9 ± 10.7%, n = 11). 3.4. Taurine induces the further potentiation after a previous saturated LTPHFS merely in lead-exposed subjects
Fig. 1. Lead exposure has opposite effects on the inductions of LTPHFS (A) and LTPTAU (B). HFS (100 Hz, 1 s) and taurine (10 mM, 30 min) were both applied on the slices of 12 control and 12 lead-exposed subjects. The recovery and enhancement of EPSP slopes were recorded at least 65 min after HFS and taurine application. For the sake of clarity, points are plotted every 2 min. Here and in subsequent figures up-arrow bar (↑) indicates the application of HFS and horizontal bar ( ) indicates the period of taurine perfusion. The inserted histograms represent the amplitudes of LTP as the mean increase in EPSP slope at 50–60 min in relation to baseline response after HFS and taurine application in the control and lead-exposed subjects (summary of the results in A and B). Asterisks indicate significant differences (P < 0.01) compared with control values.
lead exposure had no significant effect on the original potential trace during baseline measures. Fig. 1A and B (HFS and taurine, respectively) illustrate the responses to LTP induction in the 12 control and 12 lead-exposed rats. It is clear that while lead attenuates the induction of LTPHFS , it enhances the induction of LTPTAU . And, as summarized in the insert of Fig. 1A, LTPHFS amplitudes were significantly (P < 0.001) lower in lead-exposed rats (121.1 ± 8.9%) compared to controls (180.9 ± 15.9%). However, LTPTAU amplitudes were significantly higher in lead-exposed rats (199.3 ± 13.7% versus 152.3 ± 17.0%, P < 0.001). It should be noted that at the beginning of 10 mM taurine perfusion, a balance between the inhibitory action of taurine on GABAA receptors and the potentiation effect could exist. Therefore, the diminished effect of taurine on initial response suppression in the lead-exposed animal could be a result of a
del Olmo et al. [16] have demonstrated that in those synapses from which prior HFS-LTP has developed, taurine does not induce long-lasting increase in synaptic efficacy. However, the observation on lead-exposed subjects was not as such. Since GABAA receptors are not involved in taurine-induced potentiation [5], we performed a group of experiments with the presence of picrotoxin to facilitate and maximize LTP induction. A saturated LTP was induced by two 100 Hz, 1 s trains (spaced 120 s). Once the potentiation stabilized (30 min after the trains), the stimulus strength was reduced until a fEPSP of the similar pre-tetanus size was obtained. Then, after a new 10 min baseline period, 10 mM taurine was bath perfused for 30 min. When this maneuver was performed (Fig. 3A), it was observed that the fEPSP slope measured 25–29 min after trains was smaller in lead-exposed rats (128.2 ± 11.2%, n = 9) than in controls (175.5 ± 8.7%, n = 10), but following taurine withdrawal, it increased greater in lead-exposed rats (166.0 ± 11.8%) than in controls (123.1 ± 11.0%). In another set of experiments (see Fig. 3A), field potentials were evoked by increasing stimulus strength in 10 A steps. They were recorded at times as follows: before LTPHFS induction, 30 min thereafter, and 50 min after taurine withdrawal. Both control (n = 6) and lead-exposed subjects (n = 6) were examined. For each slice, fEPSP versus FV plots in the different recording duration were depicted. The representative fEPSP/FV plots from a control and a lead-treated slice were shown in Fig. 3B. It was illustrated obviously that the increase in synaptic efficacy (a
528
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
Fig. 2. The influences of lead exposure on both pre- and post-synaptic potentiation induced by taurine. (A) The traces in a represented fEPSP recorded in a slice from control animal before and after the inducement of LTPHFS during one of the experiments used for Fig. 1A. Traces in b and c were recorded in a control slice at different times (baseline, b and c) during one of the experiments used for graph C. (B) The taurine-induced increases of FV amplitude were significantly greater in lead-exposed subjects (n = 10) than in controls (n = 11). (C) To exclude the effects of FV, stimulus strength was reduced (indicated by an arrow) to size-match the FV amplitude with the baseline values after 20 min of taurine withdrawal. The fEPSP slope still remained greater in lead-exposed rats (n = 10) than in controls (n = 11).
leftward shift in the curve), which was induced by HFS, was not further changed in the controls after the taurine perfusion, while a clear increase in synaptic efficacy was evoked by taurine in the lead-exposed rats.
4. Discussion According to the previous report [5], we have known that taurine-induced synaptic potentiation consists of two long-
lasting factors: (1) the increase in the synaptic efficacy detected by a shift to the left in the fEPSP/FV curves; and (2) the enhancement of axon excitability detected by the increase of FV amplitude. The principle finding reported in this paper is that the two factors are both enhanced in the lead-exposed animals. And, it is very different from the inhibitory effect of lead-exposure on LTPHFS in which the latter factor is not involved. Since both LTPTAU components have similar extraand intra-cellular Ca2+ requirements for their maintenance
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
529
Fig. 3. After a previous LTPHFS , the subsequent taurine perfusion evoked the further potentiation of synaptic efficacy merely in the lead-exposed subjects. (A) The upper traces showed representative fEPSPs recorded from a control slice at different times (a, b, c and d on the graph) during one of the experiments used for the graph. For comparison, trace a and c were superposed, respectively, with b and c, and d. The graph plot represented the temporal evolution of fEPSP slope changes recorded from control and lead-exposed subjects. After two tetani (100 Hz, 1 s) at 2 min intervals was applied to induce a saturated LTP, at the upside down arrowhead the stimulus strength was reduced until a fEPSP of similar pre-tetanus size was obtained. Then, taurine (10 mM) was bath perfused during the time indicated by the black horizontal bar. The upside up arrowhead indicates the moment when the stimulus strength was readjusted the same as in the baseline period. (B) Representative plots of fEPSP slope vs. FV amplitude evoked in a control and a lead-exposed subject, respectively. They were recorded at times as follow: before LTP induction (䊉), 30 min thereafter () and 50 min after taurine withdrawal (). Data correspond to two experiments performed as those summarized in A but were not included in that graph.
mechanisms [16], the most probable explanation for our finding is deduced from the effects of lead on calmodulin and protein kinases. The dependence on the rise of [Ca2+ ]i is a common property for LTPTAU [16] and other types of long-lasting synaptic potentiation. And, several families of Ca2+ -sensitive kinases are involved in hippocampal LTP [12]. One mechanism by which Pb2+ appears to alert cellular physiology is replacing Ca2+ in Ca2+ -binding proteins [4], where Pb2+ may acts as an agonist, partial agonist or antagonist of the normal action of Ca2+ . For example, Pb2+ has been reported to be a fully effective agonist in activating
calmodulin [7]. However, in activating conventional Ca2+ dependent protein kinase C (PKC), Pb2+ appears to be a partial agonist, stimulating PKC at low concentration (less than about 10 nM), but to a lesser extent than Ca2+ does, inhibiting PKC at high concentrations (greater than about 100 nM), and possibly reducing the stimulatory effects of high free Ca2+ concentrations [21]. The varied effects may result from the multiple Ca2+ -binding sites of Ca2+ -activited proteins, all of which must normally be occupied by Ca2+ for full activity. When Pb2+ and Ca2+ coexist around, those sites on a single protein may be occupied solely by Ca2+ ,
530
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531
solely by Pb2+ , or possibly by a combination of both metals, depending on their absolute and relative concentrations [21]. The additive effects of Pb2+ and Ca2+ may cause important consequences for the protein activity. Some PKC- and calmodulin-stimulated proteins, such as non-NMDA receptors, type I adenylyl cyclase, plasma membrane Ca2+ -ATPase and calcineurin, are partially activated at the typical resting intracellular free Ca2+ ion concentration of 50–100 nM, and they may constantly experience enhanced stimulation by low concentrations of intracellular free Pb2+ in Pb2+ -exposed cells [9]. In our results, lead enhances the induction and maintenance of LTPTAU , while it has opposite effects on that of LTPHFS . This discrepancy may result from the different sources and lasting time in [Ca2+ ]i rise. First, to induce LTPTAU , only a small Ca2+ influx through LVACC is required and an amplification of this Ca2+ influx by Ca2+ released from intracellular stores is involved [16]. Unlike taurine, HFS cannot fully raise [Ca2+ ]i due to the NMDA receptor and voltagesensitive calcium channels being blocked by lead [2,10]. Lead cannot activate calmodulin and protein kinases without the cooperation of enough free Ca2+ [9,21]. Second, the activation of calmodulin and protein kinases may depend on the action time of the agonists. The application of taurine lasts more than a dozen minutes, which effectively activates LVACC and may cause a long-lasting [Ca2+ ]i rise [16], while [Ca2+ ]i rise disappears within seconds after HFS [13]. Third, HFS results in localized “hot spots” of free Ca2+ near the mouths of intracellular release channels or plasma membrane influx channels, in which the free Ca2+ ion concentration may reach dozens of micromolars, with the Ca2+ concentration rapidly decreasing with distance from the site of Ca2+ entry into the cytoplasm. The “hot spots” may differ from the sites of Pb2+ binding. Taurine also causes a rise in [Ca2+ ]i through the intracellular Ca2+ release channels and plasma membrane influx channels. However, the much longer duration of its effect may apply enough time for the dispersion of free Ca2+ throughout a cell, so Pb2+ and Ca2+ can effectively cooperate. Moreover, the presynaptic effect of taurine might be also involved though it is hard to get a clear conclusion by this research. In addition to the maintenance of potentiation, we should also consider the initial short-term potentiation of FV amplitude that was greatly enhanced by lead-exposure. Since intracellular application of taurine can enhance the peak Na+ current [18], Galarreta et al. have suggested a consequence of the decrease in the action potential firing threshold induced by this amino acid [5]. Moreover, chronic lead-exposure could depress the spiking activity in several types of neurons [19]. Therefore, the possibility cannot be ruled out that taurine may directly modulate the Na+ channels and remove the blockage of lead on the fiber excitability. And, a relatively greater FV increase could be expected in lead-exposed subject compared with the control. Alternatively, the lead-induced changes in the neural circuit development might also contribute to our finding.
As discussed in the previous reports [5,16], high content of taurine (5–70 mM) exists in brain cells. Also, taurine movements between extra- and intra-cellular compartments could represent the physiological framework for LTPTAU , which could also be considered as a model for several physiological and pathological situations, such as brain development and osmotic stress. If these assumptions were true, our data would reveal a more extensive interference of lead on neural system. Moreover, if taurine-induced changes in synaptic transmission were also involved in the procedure of learning and memory, our data shown in Fig. 3 would surprisingly suggest a possibility that the reinforcement of taurine might renovate the lead-induced impairment. Nevertheless, based on the present limited understanding about LTPTAU , any consideration about the physiological meaning of our finding would be very speculative. In conclusion, the chronic lead exposure increases both components accounting for LTPTAU (i.e. long-term potentiation in synaptic efficacy and FV amplitude). Moreover, though LTPTAU and LTPHFS utilize some common mechanism for the generation of long-term increases in synaptic efficacy, the quantity of calcium from different sources should be varied and thus induce different contribution to each type of LTP. Therefore, Pb2+ could cause opposite effects on LTPTAU and LTPHFS when cooperating with various [Ca2+ ]i .
Acknowledgements This work was supported by the National Basic Research program of China (No. 2002CB512907), Academic Sinica (No. KZCX3-sw-437), the National Nature Science Foundation of China (No. 30170809, 30300288, 30000039), Research Fund for the Doctoral Program of High Education (RFDP, No. 20020358053), University of Science and Technology of China (No. KB0833, KY1206).
References [1] M. Alkondon, A.C.S. Costa, V. Radhakrishnan, et al., Selective blockade of NMDA-activated channel currents may be implicated in learning deficits caused by lead, FEBS Lett. 261 (1990) 124–130. [2] G. Audesirk, T. Audesirk, Effects of in vitro lead exposure on voltage-sensitive calcium channels differ among cell types in central neurons of Lymnaea stagnalis, Neurotoxicology 10 (1989) 659–669. [3] D. Bellinger, J. Sloman, A. Leviton, et al., Low-level lead exposure and children’s cognitive function in the preschool years, Pediatrics 87 (2) (1991) 219–227. [4] C.S. Fullmer, S. Edelstein, R.H. Wasserman, Lead-binding properties of intestinal calcium-binding proteins, J. Biol. Chem. 260 (1985) 6816–6819. [5] M. Galarreta, J. Bustamante, R. Martin del Rio, et al., Taurine induces a long-lasting increase of synaptic efficacy and axon excitability in the hippocampus, J. Neurosci. 16 (1996) 92–102. [6] M.E. Gilbert, C.M. Mack, S.M. Lasley, Chronic developmental lead exposure increases the threshold for long-term potentiation in rat dentate gyrus in vivo, Brain Res. 736 (1996) 118–124.
K. Yu et al. / Brain Research Bulletin 64 (2005) 525–531 [7] E. Habermann, K. Crowell, P. Janicki, Lead and other metals can substitute for Ca2+ in calmodulin, Arch. Toxicol. 54 (1983) 61–70. [8] R.J. Huxtable, Taurine in the central nervous system and the mammalian actions of taurine, Prog. Neurobiol. 32 (1989) 471–533. [9] M. Kern, M. Wisniewski, L. Cabell, et al., Inorganic lead and calcium interact positively in activation of calmodulin, Neurotoxicology 21 (3) (2000) 353–364. [10] S.M. Lasley, M.E. Gilbert, Lead inhibits the rat N-methyl-d-aspartate receptor channel by binding to a site distinct from the zinc allosteric site, Toxicol. Appl. Pharmacol. 159 (1999) 224–233. [11] G. Lynch, M. Baudry, The biochemistry of memory: a new and specific hypothesis, Science 224 (1984) 1057–1063. [12] R.C. Malenka, R.A. Nicoll, NMDA-receptor-dependent synaptic plasticity: multiple forms and mechanisms, Trends Neurosci. 16 (1993) 521–527. [13] R.C. Malenka, R.A. Nicoll, Long-term potentiation—a decade of progress? Science 285 (1999) 1870–1874. [14] W.J. McBride, RC.A. Frederickson, Taurine as a possible inhibitory transmitter in the cerebellum, Fed Proc. 39 (1980) 2701–2705.
531
[15] S.S. Oja, P. Saransaari, Taurine as osmoregulator and neuromodulator in the brain, Metab. Brain Dis. 11 (2) (1996) 153–164. [16] N. del Olmo, M. Galarreta, J. Bustamante, et al., Taurine-induced synaptic potentiation: role of calcium and interaction with LTP, Neuropharmacology 39 (2000) 40–54. [17] D.Y. Ruan, K.F. Yan, S.Y. Ge, et al., Effects of chronic lead exposure on short-term and long-term depression in area CA1 of the rat hippocampus in vivo, Chemosphere 41 (2000) 165–171. [18] H. Satoh, Cardioprotective actions of taurine against intracellular and extracellular calcium-induced effects, Adv. Exp. Med. Biol. 359 (1994) 181–196. [19] B. Scott, J. Lew, Chronic exposure to lead causes persistent alterations in the electric membrane properties of neurons in cell culture, J. Neurobiol. 16 (6) (1985) 425–433. [20] J.A. Sturman, Taurine in development, Physiol. Rev. 73 (1993) 119–147. [21] X. Sun, X. Tian, J.L. Tomsig, et al., Analysis of differential effects of Pb2+ on protein kinase C isozymes, Toxicol. Appl. Pharmacol. 156 (1999) 40–45.