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The role of N-methyl-d -aspartate receptors in synaptic plasticity of rat visual cortex in vitro: effect of sensory experience
Neuroscience Letters 306 (2001) 149±152
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The role of N-methyl-d-aspartate receptors in synaptic plasticity of rat visual cortex in vitro: effect of sensory experience Yaghoub Fathollahi a,b,*, Mahmoud Salami c,d a Department of Physiology, School of Medical Sciences, Tarbiat Modarres University, Tehran, I. R. Iran Laboratory of Electrophysiology, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, I. R. Iran c Department of Physiology, School of Medicine, Kashan University of Medical Sciences, Kashan, I. R. Iran d Department of Physiology, School of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, I. R. Iran. b
Received 29 March 2001; received in revised form 27 April 2001; accepted 27 April 2001
Abstract We examined the role of N-methyl-d-aspartate (NMDA) receptors in synaptic plasticity of visual cortex of light (LR) and dark (DR) reared adult rats in vitro. Layer IV stimulation resulted in ®eld potentials in layer II/III, consisting of two excitatory postsynaptic potentials (EPSP) called EPSP1 and EPSP2. Tetanic stimulation induced long-term potentiation (LTP) in EPSP2 of both LR and DR visual cortices. NMDA receptor antagonist d, L-2-amino-5-phosphono-valeric acid (AP5) completely blocked the LTP of EPSP2 in DR visual cortex while it reduced slightly the extent of LTP of EPSP2 in LR ones. Another NMDA receptor antagonist ketamine blocked potentiation of EPSP1 as well as EPSP2 in both groups. Our ®ndings demonstrate that dependency of LTP on NMDA receptors and/or sensitivity of these receptors to the antagonists are different in LR and DR animals. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dark rearing; Light rearing; Long-term potentiation; N-methyl-d-aspartate receptor; Sensory experience; Visual cortex
Activity-dependent re®nement of synaptic connectivity during postnatal development of the visual cortex is thought to underlie the emergence of the adult pattern of cortical neuronal circuits [7,15]. In the developing visual cortex, activity-dependent re®nement of synaptic connectivity is thought to involve synaptic plasticity processes analogous to long-term potentiation (LTP) [22]. In the visual cortex these processes require activation of N-methyl-d-aspartate (NMDA) receptors [3,5]. Age and experience in¯uence the role of NMDA receptors in visual transmission [8,11,19]. In most of sensory structures this kind of experience-dependent synaptic plasticity is restricted to a short critical period, which can be extended by certain forms of sensory deprivation [7,16]. It has recently been shown that a threshold level of inhibition within the visual cortex may trigger, once in life, an experience-dependent critical period for circuit consolidation, which may otherwise lie dormant [9]. There are a few reports that light deprivation alters the occurrence of LTP in visual cortex responses. However, these studies present different results based on age [6] and site of stimula* Corresponding author. Department of Physiology, School of Medical Sciences, Tarbiat Modarres University, P.O. Box 14115111, Tehran, I. R. Iran. Fax: 198-21-6404680. E-mail address: [email protected] (Y. Fathollahi).
tion [17]. Therefore, the role of NMDA receptors in tetanusinduced synaptic plasticity of the visual cortex of adult rats reared under normal and dark conditions was examined. Experiments were performed on the primary visual cortex of either light (LR) or dark reared (DR) rats aged 28±42 days postnatal. LR rats were housed in standard conditions and DR ones were reared in complete darkness from birth to sacri®ce. Procedures for preparing and maintaining slices were as detailed previously [23]. Animals were initially anesthetized with ether and then decapitated. Brain was rapidly removed and placed into cold (2±48C) arti®cial cerebrospinal ¯uid (ACSF) (containing in mM: NaCl, 124; CaCl2, 2; MgCl2, 2; KH2PO4, 1.2; KCl, 4; NaHCO3, 26; glucose, 10) saturated with 95% O2 ± 5% CO2. Visual cortical slices were dissected by a vibroslicer and transferred to a humidi®ed interface-type recording chamber. Slices were perfused with ACSF at 32 ^ 28C for 1 h or more before start of experiments. Constant current pulses (25 mA, 0.1 Hz, 200 ms) were delivered through a bipolar electrode to layer IV to evoke ®eld potentials in layer II/III with two components (Fig. 1A and Fig. 2A), which were demonstrated to represent excitatory postsynaptic potentials (EPSPs) [23], called EPSP1 and EPSP2, respectively. When test responses were stable for 30 min (, 0:10 ¯uctuation), for induction of LTP, eight
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Y. Fathollahi, M. Salami / Neuroscience Letters 306 (2001) 149±152
Fig. 1. Effect of NMDA receptor antagonists on LTP of ®eld potentials recorded in LR visual cortex. (A) Representative ®eld potentials recorded from layer II/III evoked by layer IV stimulation in visual cortex of LR, LR-AP5 and LR-KET groups. Records were obtained 5 min before (baseline), and, 30 and 60 min after PBs. Each trace indicates an average of 10 consecutive records. Representative superimposed responses, which were recorded before (thick line) and 30-min after drugs application (thin line) were also shown. (B) Effects of APV and ketamine on PBs-evoked LTP of EPSP2 recorded in layer II/III of LR visual cortex. PBs of layer IV of LR visual cortex induced a signi®cant LTP in EPSP2. Notice that ketamine signi®cantly blocked the LTP production (P , 0:05, unpaired t-test, asterisk) 60 min post-PBs. Arrow indicates the time of tetanus.
episodes of primed-burst tetanic stimulation (PBs), consisting of one pulse followed 170 ms later by 10 pulses at 100 Hz,
Fig. 2. Effect of NMDA receptor antagonists on LTP of ®eld potentials recorded in DR visual cortex. (A) Representative ®eld potentials recorded from layer II/III evoked by layer IV stimulation in visual cortex of DR, DR-AP5 and DR-KET groups. Records were obtained 5 min before (baseline), and, 30 and 60 min after PBs. Each trace indicates an average of 10 consecutive records. Representative superimposed responses, which were recorded before (thick line) and 30-min after drugs application (thin line) were also shown. (B) Effects of NMDA receptor antagonists on PBs-induced LTP of EPSP2 recorded in layer II/III of DR visual cortex. PBs of layer IV of DR visual cortex triggered a considerable LTP in EPSP2 (11 out of 12 slices). Both antagonists inhibited (P , 0:01, unpaired t-test, asterisk) the LTP of EPSP2, 60 min post-PBs. Arrow indicates the time of tetanus.
were delivered at 0.1 Hz and 200 mA stimulus intensity to layer IV. In experiments where d, L-2-amino-5-phosphono-
Y. Fathollahi, M. Salami / Neuroscience Letters 306 (2001) 149±152
valeric acid [(AP5), 25±50 mM; Aldrich], a competitive NMDA receptor antagonist, and ketamine (100 mM; ParkeDavis), a non-competitive NMDA receptor antagonist, were used, the baseline recordings were obtained before and 30 min after application of the drugs. Then PBs was delivered to layer IV in the presence of the antagonists. In all experiments 30 and 60 min after tetanus, ®eld potentials of layer II/III were recorded at 25 mA stimulus intensity in the normal medium. The amplitude of EPSPs was measured from averaged waveforms for pre- and post-tetanic stimulation recordings. Slices in which the post-tetanus EPSP amplitude was at least 20% higher than the average baseline amplitude of EPSP were said to demonstrate LTP. Incidence of LTP calculated as cases showing LTP divided by the number of attempts. Repeated measures or completely randomized ANOVA were used to compare the baseline and post-PBs data within each group and between independent groups, respectively. Paired t-test was used for the absolute effects of the antagonists on baseline responses. Unpaired t-test was used to compare the independent groups for the degree of potentiation of EPSP2 60-min post-PBs. Incidence of LTP was analyzed using a non-parametric proportion analysis. Data (mean ^ SEM) obtained 1 h post-PBs were considered for statistical analysis. The LR and DR slices treated by AP5 or ketamine are abbreviated in the text and ®gures as LRAP5, DR-AP5, LR-KET and DR-KET, respectively. PBs of layer IV of LR visual cortex induced an insignificant potentiation in 7 out of 12 slices in EPSP1, but a robust LTP in 8 out of 12 slices in EPSP2 (212.81 ^ 47.88%; F2;22 8, P , 0:01, ANOVA) (Fig. 1). In DR slices, PBs of layer IV elicited LTP in 7 out of 12 slices in EPSP1 and in 11 out of 12 slices in EPSP2 (Fig. 2). However, the amount of potentiation was signi®cant only in EPSP2 (197.86 ^ 23.51%; F2;22 18, P , 0:01, ANOVA). In DR rats the enhancement of EPSP1 amplitude was larger, and that of EPSP2 was smaller than in LR rats. In both LR and DR animals, the magnitude of potentiation of EPSP2 on average was greater than that of EPSP1. The amplitude changes (i.e. after 60 min for EPSP2) for LR and DR were not statistically signi®cant. Incubation of the LR slices with ACSF containing 25±50 mM AP5 signi®cantly suppressed the amplitude of EPSP1 (76.4 ^ 6.67% of pre-AP5 values, P , 0:05) but did not in¯uence that of EPSP2 (94.84 ^ 5% of pre-AP5 values). PBs of layer IV triggered LTP in 3 out of 6 slices in EPSP1 and there was no signi®cant difference between LR and LRAP5 groups (115.23 ^ 9.27% vs. 117.96 ^ 15.96%; F1;35 1:57, P , 0:219, ANOVA). Although AP5 decreased the potentiation level of EPSP2, 5 out of 6 slices expressed LTP (144.83 ^ 28.35%). But there was no significant difference (F1;35 0:3, P 0:58; ANOVA, Fig. 1B) between LR and LR-AP5 slices. LTP was also induced in the presence of 100 mM-AP5. Perfusion of the DR slices with medium containing 25 to 50 mM AP5 did not change the amplitude of EPSP1 (99.46 ^ 4% of pre-AP5 values) and EPSP2 (90.2 ^ 8%
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of pre-AP5 values). PBs of layer IV in the presence of AP5 enhanced the amplitude of EPSP1 in 3 out of 6 slices (135.52 ^ 9.84% vs. 119.22 ^ 16.15%), but the difference between DR and DR-AP5 groups was not signi®cant. AP5 completely blocked the LTP of EPSP2 (101.09 ^ 18.08%; F1;32 8:93, P , 0:005; ANOVA, Fig. 2B). The incidence of LTP of EPSP2 for DR-APV slices (0.3) was markedly different (P , 0:05) from that of LR-APV group (0.83). Our results demonstrated that the NMDA receptor antagonist AP5 did not in¯uence the LTP of both EPSPs in adult LR visual cortex. Similarly, Aroniadou and Teyler showed that AP5 decreases the baseline amplitude and LTP of EPSP2 in young (18±25 days old) [1] but not in adult (52±85 days old) [2] rats. It has been reported that binding sites for the NMDA receptor antagonist MK-801 decreases in the adult visual cortex [14]. Also it is known that during the development of glutamatergic transmission in sensory cortex, change in the composition of NMDA receptors shortens the receptor currents [10] and reduces contribution of NMDA receptors to synaptic currents [12]. The present data and those reported previously [2] demonstrate that an NMDA receptor independent mechanism is involved, at least partially, in the induction of LTP in adult visual cortex. However, Komatsu et al. found such an NMDA receptorindependent LTP in the kitten visual cortex [18]. On the other hand, while AP5 was ineffective on the baseline level of EPSP2 in both groups, it inhibited LTP of EPSP2 only in DR rats, because the probability of LTP in DR-APV was markedly different from that of LR-APV. Fox et al. reported that visual deprivation delays loss of NMDA receptor function in the visual cortex [11]. If so, it would be expectable to observe a substantial role for NMDA receptors in LTP of adult DR visual cortex. To further con®rm the role of NMDA receptors in LTP of visual cortical responses, we assessed occurrence of LTP of EPSPs in both groups using another NMDA receptor antagonist ketamine. In LR rats, ketamine prevented the potentiation rate of EPSP1 (95.40 ^ 6.66%; F1;49 10:5, P , 0:05, ANOVA) and decreased the LTP of EPSP2 (123.66 ^ 16.82; F1;51 8:39, P , 0:05, ANOVA), (Fig. 1B). Also, preincubation of the DR slices with 100 mM ketamine reduced the degree of potentiation of both EPSP1 (110.84 ^ 8.54%; F1;34 6:7, P , 0:05, ANOVA) and EPSP2 (110.93 ^ 4.90; F1;37 14:5, P , 0:01, ANOVA), (Fig. 2B). Ketamine antagonized LTP of EPSP1. This effect of ketamine may, in part, be attributed to its effect on EPSP1b (a component of EPSP1 mediated by NMDA receptors). Similarly, it has been reported that AP5 is also able to prevent LTP of EPSP1 [4]. Considering that a component of EPSP1 (i.e. EPSP1a) is non-NMDA receptor dependent, it seems likely that ketamine blocked nonNMDA as well as NMDA receptors, although this remains to be elucidated. Gonzales et al. [13] demonstrated that quisqaulate-stimulated (but not kainate) responses were more sensitive to ketamine than were NMDA-stimulated responses.
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Taken together, these results raise the possibility that, at least in part, LTP of response components in both groups could be mediated by NMDA receptors but, in LR rats, these receptors are not very sensitive to AP5. Dark rearing changes the developmental increase of the NR2A subunit, the subunit that may affect the receptor sensitivity to ligands, of NMDA receptor in postnatal age so that NR2A/NR2B is higher in LR than in DR animals [21]. Therefore, it seems likely that dark rearing retains NMDA receptors sensitive to AP5. Another possibility is that inhibitory circuits might be less active in DR visual cortex. While Gordon et al. [14] demonstrated that dark rearing decreased muscimol (a GABAA receptor agonist) binding site, Mower et al. [20] believed that postnatal development of GABA neurons and receptors occurs normally in the absence of visual inputs. It has also been reported that GABA receptors determine critical periods and in fact NMDA receptors maturation may not be the decisive factor [9]. Brie¯y, our ®ndings show that the dependency of LTP on NMDA receptors and/or the sensitivity of these receptors to the antagonists are different between LR and DR animals. These results are consistent with the ®ndings that the composition and action of NMDA receptors alters as a function of age and sensory experience. [1] Aroniadou, V.A. and Teyler, T.J., The role of NMDA receptors in long-term potentiation (LTP) and depression (LTD) in rat visual cortex, Brain Res., 562 (1991) 136±143. [2] Aroniadou, V.A. and Teyler, T.J., Induction of NMDA receptor-independent long-term potentiation (LTP) in visual cortex, Brain Res., 548 (1992) 169±173. [3] Artola, A. and Singer, W., Long-term potentiation and NMDA receptors in rat visual cortex, Nature, 330 (1987) 649±652. [4] Artola, A. and Singer, W., The involvement of N-methyl-Daspartate receptors in induction and maintenance of longterm potentiation in rat visual cortex, Eur. J. Neurosci., 12 (1990) 254±269. [5] Bear, M.F., Kleischmidt, A., Gu, Q. and Singer, W., Disruption of experience-dependent synaptic modi®cations in striate cortex by infusion of an NMDA receptor antagonist, J. Neurosci., 10 (1990) 88±92. [6] Berry, R.L., Perkins, A.T. and Teyler, T.J., Visual deprivation decreases long-term potentiation in visual cortical slices, Brain Res., 628 (1993) 99±104. [7] Binns, K.E., Turner, J.P. and Salt, T.E., Visual experience alters the molecular pro®le of NMDA-receptor-mediated sensory transmission, Eur. J. Neurosci., 11 (1999) 1101± 1104. [8] Carmignoto, G. and Vicini, S., Activity-dependent decrease
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in NMDA receptor responses during development of the visual cortex, Science, 258 (1992) 1007±1011. Fagiolini, M. and Hensch, T.K., Inhibitory threshold for critical-period activation in primary visual cortex, Nature, 404 (2000) 183±186. Flint, A.C., Maisch, U.S., Weishaupt, J.H., Kreigstein, A.R. and Monyer, H., NR2a sub-unit expression shortens NMDA receptor synaptic currents in developing neocortex, J. Neurosci., 17 (1997) 2469±2476. Fox, K., Daw, N., Sato, H. and Czepita, D., Dark-rearing delays the loss of NMDA-receptor function in kitten visual cortex, Nature, 350 (1991) 342±344. Fox, K., Henley, J. and Issac, J., Experience-dependent development of NMDA receptor transmission, Nat. Neurosci., 2 (1999) 297±299. Gonzales, J.M., Loeb, A.L., Reichard, P.S. and Irvine, S., Ketamine inhibits glutamate-, N-methyl-D-aspartate-, and quisqualate-stimulated cGMP production in cultured cerebral neurons, Anesthesiology, 82 (1995) 205±213. Gordon, B., Daw, N. and Parkinson, D., The effect of age on binding of MK-801 in the cat visual cortex, Dev. Brain Res., 62 (1991) 61±67. Katz, L.C. and Shatz, C.J., Synaptic activity and the construction of cortical circuits, Science, 274 (1996) 1133± 1138. Kirkwood, A., lee, H.K. and Bear, M.F., Co-regulation of long-term potentiation and experience-dependent synaptic plasticity in visual cortex by age and experience, Nature, 375 (1995) 328±331. Kirkwood, A., Riolt, M.G. and Bear, M.F., Experience-dependent modi®cation of synaptic plasticity in visual cortex, Nature, 381 (1996) 526±528. Komatsu, Y., Nakajima, S. and Toyama, K., Induction of long-term potentiation without participation of N-methylD-aspartate receptors in kitten visual cortex, J. Neurophysiol., 65 (1991) 20±32. Monyer, H., Burnashev, N., Laurie, D.J., Sakmann, B. and Seeburg, P.H., Developmental and regional expression in the rat brain and functional properties of four NMDA receptors, Neuron, 12 (1994) 529±540. Mower, G.D., Rustad, R. and White, W.F., Quantitative comparisons of gamma-aminobutyric acid neurons and receptors in the visual cortex of normal and dark-reared cats, J. Comp. Neurol., 272 (1988) 293±302. Quinlan, E.M., Philpot, B.D., Huganir, R.L. and Bear, M.F., Rapid, experience-dependent expression of synaptic NMDA receptor in visual cortex in vivo, Nat. Neurosci., 21 (1999) 352±357. Rumpel, S., Hatt, H. and Gottmann, K., Silent synapses in the developing rat visual cortex: evidence for postsynaptic expression of synaptic plasticity, J. Neurosci., 18 (1998) 8863±8874. Salami, M., Fathollahi, Y., Esteky, H., Motamedi, F. and Atapour, N., Effects of ketamine on synaptic transmission and long-term potentiation in layer II/III of rat visual cortex in vitro, Eur. J. Pharmacol., 390 (2000) 287±293.
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The role of N-methyl-d-aspartate receptors in synaptic plasticity of rat visual cortex in vitro: effect of sensory experience Yaghoub Fathollahi a,b,*, Mahmoud Salami c,d a Department of Physiology, School of Medical Sciences, Tarbiat Modarres University, Tehran, I. R. Iran Laboratory of Electrophysiology, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, I. R. Iran c Department of Physiology, School of Medicine, Kashan University of Medical Sciences, Kashan, I. R. Iran d Department of Physiology, School of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, I. R. Iran. b
Received 29 March 2001; received in revised form 27 April 2001; accepted 27 April 2001
Abstract We examined the role of N-methyl-d-aspartate (NMDA) receptors in synaptic plasticity of visual cortex of light (LR) and dark (DR) reared adult rats in vitro. Layer IV stimulation resulted in ®eld potentials in layer II/III, consisting of two excitatory postsynaptic potentials (EPSP) called EPSP1 and EPSP2. Tetanic stimulation induced long-term potentiation (LTP) in EPSP2 of both LR and DR visual cortices. NMDA receptor antagonist d, L-2-amino-5-phosphono-valeric acid (AP5) completely blocked the LTP of EPSP2 in DR visual cortex while it reduced slightly the extent of LTP of EPSP2 in LR ones. Another NMDA receptor antagonist ketamine blocked potentiation of EPSP1 as well as EPSP2 in both groups. Our ®ndings demonstrate that dependency of LTP on NMDA receptors and/or sensitivity of these receptors to the antagonists are different in LR and DR animals. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dark rearing; Light rearing; Long-term potentiation; N-methyl-d-aspartate receptor; Sensory experience; Visual cortex
Activity-dependent re®nement of synaptic connectivity during postnatal development of the visual cortex is thought to underlie the emergence of the adult pattern of cortical neuronal circuits [7,15]. In the developing visual cortex, activity-dependent re®nement of synaptic connectivity is thought to involve synaptic plasticity processes analogous to long-term potentiation (LTP) [22]. In the visual cortex these processes require activation of N-methyl-d-aspartate (NMDA) receptors [3,5]. Age and experience in¯uence the role of NMDA receptors in visual transmission [8,11,19]. In most of sensory structures this kind of experience-dependent synaptic plasticity is restricted to a short critical period, which can be extended by certain forms of sensory deprivation [7,16]. It has recently been shown that a threshold level of inhibition within the visual cortex may trigger, once in life, an experience-dependent critical period for circuit consolidation, which may otherwise lie dormant [9]. There are a few reports that light deprivation alters the occurrence of LTP in visual cortex responses. However, these studies present different results based on age [6] and site of stimula* Corresponding author. Department of Physiology, School of Medical Sciences, Tarbiat Modarres University, P.O. Box 14115111, Tehran, I. R. Iran. Fax: 198-21-6404680. E-mail address: [email protected] (Y. Fathollahi).
tion [17]. Therefore, the role of NMDA receptors in tetanusinduced synaptic plasticity of the visual cortex of adult rats reared under normal and dark conditions was examined. Experiments were performed on the primary visual cortex of either light (LR) or dark reared (DR) rats aged 28±42 days postnatal. LR rats were housed in standard conditions and DR ones were reared in complete darkness from birth to sacri®ce. Procedures for preparing and maintaining slices were as detailed previously [23]. Animals were initially anesthetized with ether and then decapitated. Brain was rapidly removed and placed into cold (2±48C) arti®cial cerebrospinal ¯uid (ACSF) (containing in mM: NaCl, 124; CaCl2, 2; MgCl2, 2; KH2PO4, 1.2; KCl, 4; NaHCO3, 26; glucose, 10) saturated with 95% O2 ± 5% CO2. Visual cortical slices were dissected by a vibroslicer and transferred to a humidi®ed interface-type recording chamber. Slices were perfused with ACSF at 32 ^ 28C for 1 h or more before start of experiments. Constant current pulses (25 mA, 0.1 Hz, 200 ms) were delivered through a bipolar electrode to layer IV to evoke ®eld potentials in layer II/III with two components (Fig. 1A and Fig. 2A), which were demonstrated to represent excitatory postsynaptic potentials (EPSPs) [23], called EPSP1 and EPSP2, respectively. When test responses were stable for 30 min (, 0:10 ¯uctuation), for induction of LTP, eight
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 89 4- 8
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Fig. 1. Effect of NMDA receptor antagonists on LTP of ®eld potentials recorded in LR visual cortex. (A) Representative ®eld potentials recorded from layer II/III evoked by layer IV stimulation in visual cortex of LR, LR-AP5 and LR-KET groups. Records were obtained 5 min before (baseline), and, 30 and 60 min after PBs. Each trace indicates an average of 10 consecutive records. Representative superimposed responses, which were recorded before (thick line) and 30-min after drugs application (thin line) were also shown. (B) Effects of APV and ketamine on PBs-evoked LTP of EPSP2 recorded in layer II/III of LR visual cortex. PBs of layer IV of LR visual cortex induced a signi®cant LTP in EPSP2. Notice that ketamine signi®cantly blocked the LTP production (P , 0:05, unpaired t-test, asterisk) 60 min post-PBs. Arrow indicates the time of tetanus.
episodes of primed-burst tetanic stimulation (PBs), consisting of one pulse followed 170 ms later by 10 pulses at 100 Hz,
Fig. 2. Effect of NMDA receptor antagonists on LTP of ®eld potentials recorded in DR visual cortex. (A) Representative ®eld potentials recorded from layer II/III evoked by layer IV stimulation in visual cortex of DR, DR-AP5 and DR-KET groups. Records were obtained 5 min before (baseline), and, 30 and 60 min after PBs. Each trace indicates an average of 10 consecutive records. Representative superimposed responses, which were recorded before (thick line) and 30-min after drugs application (thin line) were also shown. (B) Effects of NMDA receptor antagonists on PBs-induced LTP of EPSP2 recorded in layer II/III of DR visual cortex. PBs of layer IV of DR visual cortex triggered a considerable LTP in EPSP2 (11 out of 12 slices). Both antagonists inhibited (P , 0:01, unpaired t-test, asterisk) the LTP of EPSP2, 60 min post-PBs. Arrow indicates the time of tetanus.
were delivered at 0.1 Hz and 200 mA stimulus intensity to layer IV. In experiments where d, L-2-amino-5-phosphono-
Y. Fathollahi, M. Salami / Neuroscience Letters 306 (2001) 149±152
valeric acid [(AP5), 25±50 mM; Aldrich], a competitive NMDA receptor antagonist, and ketamine (100 mM; ParkeDavis), a non-competitive NMDA receptor antagonist, were used, the baseline recordings were obtained before and 30 min after application of the drugs. Then PBs was delivered to layer IV in the presence of the antagonists. In all experiments 30 and 60 min after tetanus, ®eld potentials of layer II/III were recorded at 25 mA stimulus intensity in the normal medium. The amplitude of EPSPs was measured from averaged waveforms for pre- and post-tetanic stimulation recordings. Slices in which the post-tetanus EPSP amplitude was at least 20% higher than the average baseline amplitude of EPSP were said to demonstrate LTP. Incidence of LTP calculated as cases showing LTP divided by the number of attempts. Repeated measures or completely randomized ANOVA were used to compare the baseline and post-PBs data within each group and between independent groups, respectively. Paired t-test was used for the absolute effects of the antagonists on baseline responses. Unpaired t-test was used to compare the independent groups for the degree of potentiation of EPSP2 60-min post-PBs. Incidence of LTP was analyzed using a non-parametric proportion analysis. Data (mean ^ SEM) obtained 1 h post-PBs were considered for statistical analysis. The LR and DR slices treated by AP5 or ketamine are abbreviated in the text and ®gures as LRAP5, DR-AP5, LR-KET and DR-KET, respectively. PBs of layer IV of LR visual cortex induced an insignificant potentiation in 7 out of 12 slices in EPSP1, but a robust LTP in 8 out of 12 slices in EPSP2 (212.81 ^ 47.88%; F2;22 8, P , 0:01, ANOVA) (Fig. 1). In DR slices, PBs of layer IV elicited LTP in 7 out of 12 slices in EPSP1 and in 11 out of 12 slices in EPSP2 (Fig. 2). However, the amount of potentiation was signi®cant only in EPSP2 (197.86 ^ 23.51%; F2;22 18, P , 0:01, ANOVA). In DR rats the enhancement of EPSP1 amplitude was larger, and that of EPSP2 was smaller than in LR rats. In both LR and DR animals, the magnitude of potentiation of EPSP2 on average was greater than that of EPSP1. The amplitude changes (i.e. after 60 min for EPSP2) for LR and DR were not statistically signi®cant. Incubation of the LR slices with ACSF containing 25±50 mM AP5 signi®cantly suppressed the amplitude of EPSP1 (76.4 ^ 6.67% of pre-AP5 values, P , 0:05) but did not in¯uence that of EPSP2 (94.84 ^ 5% of pre-AP5 values). PBs of layer IV triggered LTP in 3 out of 6 slices in EPSP1 and there was no signi®cant difference between LR and LRAP5 groups (115.23 ^ 9.27% vs. 117.96 ^ 15.96%; F1;35 1:57, P , 0:219, ANOVA). Although AP5 decreased the potentiation level of EPSP2, 5 out of 6 slices expressed LTP (144.83 ^ 28.35%). But there was no significant difference (F1;35 0:3, P 0:58; ANOVA, Fig. 1B) between LR and LR-AP5 slices. LTP was also induced in the presence of 100 mM-AP5. Perfusion of the DR slices with medium containing 25 to 50 mM AP5 did not change the amplitude of EPSP1 (99.46 ^ 4% of pre-AP5 values) and EPSP2 (90.2 ^ 8%
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of pre-AP5 values). PBs of layer IV in the presence of AP5 enhanced the amplitude of EPSP1 in 3 out of 6 slices (135.52 ^ 9.84% vs. 119.22 ^ 16.15%), but the difference between DR and DR-AP5 groups was not signi®cant. AP5 completely blocked the LTP of EPSP2 (101.09 ^ 18.08%; F1;32 8:93, P , 0:005; ANOVA, Fig. 2B). The incidence of LTP of EPSP2 for DR-APV slices (0.3) was markedly different (P , 0:05) from that of LR-APV group (0.83). Our results demonstrated that the NMDA receptor antagonist AP5 did not in¯uence the LTP of both EPSPs in adult LR visual cortex. Similarly, Aroniadou and Teyler showed that AP5 decreases the baseline amplitude and LTP of EPSP2 in young (18±25 days old) [1] but not in adult (52±85 days old) [2] rats. It has been reported that binding sites for the NMDA receptor antagonist MK-801 decreases in the adult visual cortex [14]. Also it is known that during the development of glutamatergic transmission in sensory cortex, change in the composition of NMDA receptors shortens the receptor currents [10] and reduces contribution of NMDA receptors to synaptic currents [12]. The present data and those reported previously [2] demonstrate that an NMDA receptor independent mechanism is involved, at least partially, in the induction of LTP in adult visual cortex. However, Komatsu et al. found such an NMDA receptorindependent LTP in the kitten visual cortex [18]. On the other hand, while AP5 was ineffective on the baseline level of EPSP2 in both groups, it inhibited LTP of EPSP2 only in DR rats, because the probability of LTP in DR-APV was markedly different from that of LR-APV. Fox et al. reported that visual deprivation delays loss of NMDA receptor function in the visual cortex [11]. If so, it would be expectable to observe a substantial role for NMDA receptors in LTP of adult DR visual cortex. To further con®rm the role of NMDA receptors in LTP of visual cortical responses, we assessed occurrence of LTP of EPSPs in both groups using another NMDA receptor antagonist ketamine. In LR rats, ketamine prevented the potentiation rate of EPSP1 (95.40 ^ 6.66%; F1;49 10:5, P , 0:05, ANOVA) and decreased the LTP of EPSP2 (123.66 ^ 16.82; F1;51 8:39, P , 0:05, ANOVA), (Fig. 1B). Also, preincubation of the DR slices with 100 mM ketamine reduced the degree of potentiation of both EPSP1 (110.84 ^ 8.54%; F1;34 6:7, P , 0:05, ANOVA) and EPSP2 (110.93 ^ 4.90; F1;37 14:5, P , 0:01, ANOVA), (Fig. 2B). Ketamine antagonized LTP of EPSP1. This effect of ketamine may, in part, be attributed to its effect on EPSP1b (a component of EPSP1 mediated by NMDA receptors). Similarly, it has been reported that AP5 is also able to prevent LTP of EPSP1 [4]. Considering that a component of EPSP1 (i.e. EPSP1a) is non-NMDA receptor dependent, it seems likely that ketamine blocked nonNMDA as well as NMDA receptors, although this remains to be elucidated. Gonzales et al. [13] demonstrated that quisqaulate-stimulated (but not kainate) responses were more sensitive to ketamine than were NMDA-stimulated responses.
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Taken together, these results raise the possibility that, at least in part, LTP of response components in both groups could be mediated by NMDA receptors but, in LR rats, these receptors are not very sensitive to AP5. Dark rearing changes the developmental increase of the NR2A subunit, the subunit that may affect the receptor sensitivity to ligands, of NMDA receptor in postnatal age so that NR2A/NR2B is higher in LR than in DR animals [21]. Therefore, it seems likely that dark rearing retains NMDA receptors sensitive to AP5. Another possibility is that inhibitory circuits might be less active in DR visual cortex. While Gordon et al. [14] demonstrated that dark rearing decreased muscimol (a GABAA receptor agonist) binding site, Mower et al. [20] believed that postnatal development of GABA neurons and receptors occurs normally in the absence of visual inputs. It has also been reported that GABA receptors determine critical periods and in fact NMDA receptors maturation may not be the decisive factor [9]. Brie¯y, our ®ndings show that the dependency of LTP on NMDA receptors and/or the sensitivity of these receptors to the antagonists are different between LR and DR animals. These results are consistent with the ®ndings that the composition and action of NMDA receptors alters as a function of age and sensory experience. [1] Aroniadou, V.A. and Teyler, T.J., The role of NMDA receptors in long-term potentiation (LTP) and depression (LTD) in rat visual cortex, Brain Res., 562 (1991) 136±143. [2] Aroniadou, V.A. and Teyler, T.J., Induction of NMDA receptor-independent long-term potentiation (LTP) in visual cortex, Brain Res., 548 (1992) 169±173. [3] Artola, A. and Singer, W., Long-term potentiation and NMDA receptors in rat visual cortex, Nature, 330 (1987) 649±652. [4] Artola, A. and Singer, W., The involvement of N-methyl-Daspartate receptors in induction and maintenance of longterm potentiation in rat visual cortex, Eur. J. Neurosci., 12 (1990) 254±269. [5] Bear, M.F., Kleischmidt, A., Gu, Q. and Singer, W., Disruption of experience-dependent synaptic modi®cations in striate cortex by infusion of an NMDA receptor antagonist, J. Neurosci., 10 (1990) 88±92. [6] Berry, R.L., Perkins, A.T. and Teyler, T.J., Visual deprivation decreases long-term potentiation in visual cortical slices, Brain Res., 628 (1993) 99±104. [7] Binns, K.E., Turner, J.P. and Salt, T.E., Visual experience alters the molecular pro®le of NMDA-receptor-mediated sensory transmission, Eur. J. Neurosci., 11 (1999) 1101± 1104. [8] Carmignoto, G. and Vicini, S., Activity-dependent decrease
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