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Tetraethylammonium-induced long-term potentiation in layer V horizontal connections of rat motor cortex
Neuroscience Letters 298 (2001) 37±40
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Tetraethylammonium-induced long-term potentiation in layer V horizontal connections of rat motor cortex Przemyslaw Jagodzinski, Grzegorz Hess* Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Krakow, Poland Received 8 November 2000; received in revised form 27 November 2000; accepted 27 November 2000
Abstract The possibility for the induction of long-lasting synaptic plasticity by the potassium channel blocker tetraethylammonium (TEA) was investigated in adult rat motor cortex in vitro. Brief application of TEA (25 mM) resulted in a long-term potentiation (LTPK) of ®eld potentials evoked in layer V intralaminar connections by 59 ^ 17%. This effect could be prevented by preincubation with nifedipine, a voltage-dependent calcium channel blocker (20 mM), but not by Nmethyl-d-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV, 100 mM). LTPK induction resulted in a smaller relative potentiation of a response to the second pulse of a pair (60 ms interpulse interval). These results indicate a potential of layer V horizontal connections for a NMDA receptor-independent form of persistent synaptic enhancement. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Synaptic plasticity; Brain slice; K 1 channel; Ca 21 channel; Paired-pulse; Adult rat
Horizontally-oriented intracortical connections have been proposed to serve as a substrate for plastic reorganizations of adult motor cortex [16]. N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) of synaptic ef®cacy is a candidate mechanism of these reorganizations in mammalian species ranging from rats to humans [6,11]. In slice preparations of the hippocampus, where LTP mechanisms have been extensively investigated, apart from NMDA receptor-dependent LTP another form of LTP has been described which can be induced under conditions of NMDA receptor blockade by a very highfrequency afferent activation. This form of LTP is induced by the in¯ux of Ca 21 ions through voltage-dependent Ca 21 channels (VDCCs) of l-type [9]. Similarly, a transient exposure of hippocampal slices to a potassium channel blocker tetraethylammonium (TEA) results in a long-lasting increase of synaptic responses, referred to as LTPK, which depends on the Ca 21 in¯ux into post-synaptic cells through l-type VDCCs [1,13,20]. Neocortical VDCC-dependent forms of LTP have not been well explored. VDCCs are involved in LTP induction in layer II/III of the motor cortex of immature rats [3], however, since a potential for synaptic plasticity diminishes * Corresponding author. Tel.: 148-12-633-6377, ext. 2602; fax: 148-12-634-3716. E-mail address: [email protected] (G. Hess).
markedly with age, at least in the sensory cortices after the critical period [8], it is important to determine whether adult neocortex expresses this property. The induction of VDCCs-dependent LTPK has been reported for synaptic connections terminating on layer II/III pyramidal neurons of rat frontal cortex [19] but it is not known if this form of LTP is operable in layer V connections. To test this possibility we investigated the effects of TEA on synaptic transmission in layer V horizontal connections of the motor cortex (MI). Brain slices containing a part of MI were prepared from young adult female and male rats aged 6±9 weeks. Animals were deeply anesthetized with sodium pentobarbital applied intraperitoneally and decapitated. The brain was removed and immersed in chilled (7± 88C) arti®cial cerebrospinal ¯uid (ACSF) of the following composition (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3 and 15 d-glucose, bubbled with 95% O2-5% CO2. Parasagittal slices (450 mm) were cut at the distance of 2.0±3.0 mm from the brain midline using a vibrating microtome and transferred to a ¯uid-gas interface chamber perfused (1 ml/min) with warmed ACSF (34:5 ^ 0:58C). The humidi®ed atmosphere over the slices was saturated with a mixture of 95% O2±5% CO2. Concentric bipolar platinum/stainless steel stimulating electrodes were placed 1100±1300 mm below the cortical surface to activate ®bers running within layer V [12]. Constant-current or constant-voltage, 0.2 ms pulses were
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 0) 01 71 5- 8
38
P. Jagodzinski, G. Hess / Neuroscience Letters 298 (2001) 37±40
delivered at 0.033 Hz. Test stimulus intensity was adjusted to evoke a half-maximum synaptic response based on inputoutput curves. Field potentials were recorded using glass micropipettes ®lled with 0.25 M NaCl (3±6 MV) from sites located approx. 500 mm from the stimulation sites. Data were ampli®ed, ®ltered (0.1±500 Hz), acquired at a 10 kHz sampling rate and analyzed on- and off-line. The amplitude of the initial negative component of the response was used as a measure of the population excitatory synaptic current because this potential re¯ects monosynaptic current sink [2,12]. Intracellular recordings were obtained using sharp microelectrodes ®lled with 3 M K-acetate (60±80 MV). Only cells having a resting membrane potential greater than 270 mV, input resistance $20 MV and overshooting action potentials were recorded. Tetraethylammonium chloride, nifedipine and d,l-2-amino-5-phosphonovalerate (Sigma) were applied in the bathing ACSF. The results are presented as means ^ SEM unless noted. The exposure of slices to 25 mM TEA for 11 min resulted typically (nine of 11 slices) in a long-term increase of ®eld potentials (LTPK) evoked in layer V intralaminar connections. LTPK developed gradually over about 30 min after termination of TEA application and then remained stable for more than 1.5 h. In two slices responses returned to nearcontrol values. Fig. 1A shows that at this timepoint the average ®eld potential peak amplitude was increased to 159 ^ 17% of baseline (n 11). Layer V LTPK was not dependent on stimulus-evoked synaptic activity, since in the absence of testing electrical stimuli the long-term
response increase of comparable magnitude could be produced when tested 90 min after TEA application (not shown). Intracellular recordings from four layer V regularspiking neurons [18] demonstrated that TEA induced a similar increase of post-synaptic potentials (Fig. 1B). Among the set of neurons the post-synaptic potential peak amplitude increased by 22±64%. Consistent with previous studies [1,19] the effect was not due to permanently increased excitability of these cells (Fig. 1C). It has been reported that the hippocampal LTPK may consist of two components±one dependent on the activation of l-type calcium channels while the other, on NMDA receptors [10,14]. To test the involvement of both mechanisms in LTPK of the motor cortex in a next set of experiments TEA was applied in the presence of nifedipine, a calcium channel blocker. Pretreatment of the slices with 20 mM nifedipine did not markedly change recorded ®eld potentials, however, it blocked the long-term effect of TEA application. As illustrated in Fig. 2A, after a transient TEAinduced increase ®eld potential amplitude returned to 108 ^ 5% of control values (n 11) within 70 min. In contrast, the possibility for LTPK induction was not in¯uenced by d,l-2-amino-5-phosphonovalerate (APV, 100 mM), an antagonist of NMDA receptors. Among the four tested slices, in agreement with earlier ®ndings [12], the addition of APV to the ACSF resulted in a small reduction of late part of the ®eld potential beginning after the peak of the response. As shown in Fig. 2B, the application of TEA in the continuous presence of APV resulted in an increase of
Fig. 1. LTPK in layer V horizontal connections. (A1) Mean effect (^SEM, n 11) of 25 mM TEA applied for 11 min (bar) on intralaminar layer V ®eld potential amplitude. (A2) Superpositions of averages of ten responses recorded at times indicated in A1 during a representative experiment. Note a prolonged response timecourse at time 3, most likely due to enhanced polysynaptic transmission. (B1) LTPK of post-synaptic potentials recorded from a representative layer V neuron. Broken lines represent ^2 SD over baseline period. (B2) Superposition of averages (n 10) of post-synaptic potentials recorded as indicated in B1. (C) Response of the cell to a threshold, 400 ms long depolarizing current pulse applied before (control, 0.5 nA) and after (90 min, 0.6 nA) the termination of TEA application.
P. Jagodzinski, G. Hess / Neuroscience Letters 298 (2001) 37±40
39
Fig. 2. LTPK is blocked by nifedipine but not by APV. (A) Summary of experiments (^SEM, n 11) in which TEA was applied in the presence of nifedipine. Bars indicate the duration of drug applications. (B) Summary of experiments (^SEM, n 4) in which APV was present throughout the experiment and TEA was applied as indicated by a bar.
response to 152 ^ 8% of control values (n 4) 70 min after termination of TEA application. This result indicates that LTPK may be induced and expressed entirely by nonNMDA receptor-based synaptic transmission. To get further insight into LTPK mechanism, paired-pulse evoked ®eld potentials were analyzed. As demonstrated in Fig. 3, during the control period paired-pulse stimulation (60 ms interpulse interval) of layer V horizontal connections resulted in a slight facilitation of the response to the second pulse. On average its amplitude in relation to that evoked by the ®rst pulse (R2/R1) was 1:054 ^ 0:027 (n 8). However, after the induction of LTPK the R2/R1 ratio was reduced to 0:916 ^ 0:049 (P , 0:001, paired t-test), indicating a smaller degree of potentiation of the response to the second pulse. Our results demonstrate that the transient exposure of the slices containing a part of the motor cortex to 25 mM TEA results in a long-term increase of ®eld potentials evoked in layer V intralaminar connections. Earlier in vivo studies on adult motor cortical synaptic plasticity suggested that there may be layer differences in the potential for LTP. It has been noted that although some inputs to layer V pyramidal neurons may undergo activity-dependent long-lasting potentiation under speci®c experimental conditions [4,17], LTP is induced more readily in layers II±III [15]. The present results
complement the data provided by Pelletier and Hablitz [19] in showing that LTPK may be readily induced in deep layers of the rat neocortex. Similarly to that study and to the hippocampal LTPK [1], we note that layer V LTPK is characterized by a gradual increase of synaptic responses after TEA withdrawal. The mechanism of layer V LTPK induction is similar to that of layer II/III [19], since in both cases it is dependent on nifedipine-sensitive VDCCs, presumably of l-type, but not on NMDA receptors and does not require stimulationevoked synaptic activity. The involvement of NMDA receptors in LTPK induction in the hippocampus has been a matter of discussion. While the initial studies suggested that LTPK is entirely dependent on VDCCs [1,13], it was later demonstrated that it may have a NMDA receptor-dependent component [10] which required presynaptic stimulation [14]. The present results con®rm a lack of the involvement of NMDA receptors in neocortical LTPK [19], although it can not be excluded that NMDA receptors are involved in short-lasting effects of TEA application [14]. It has been reported that in the hippocampus LTPK does not interact with paired-pulse facilitation [13]. The present data, however, demonstrate a change in the relationship between responses evoked by two closely spaced pulses. Since the moderate electrical stimulation of layer V engages
Fig. 3. Interaction between LTPK and paired-pulse plasticity. (A) The paired-pulse ratio (amplitude of the second response/amplitude of the ®rst response, R2/R1) before (con) and after (post) LTPK induction (^SEM, n 8). (B) Superpositions of averages of ®eld potentials evoked by paired pulses before (thin line) and after (thick line) LTPK induction at 45 mA in a representative experiment (upper graph). Lower graph shows a superposition of above traces which were scaled to match the amplitude of the response to the ®rst pulse (R1). Note a difference in the amplitude of the second response (R2).
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P. Jagodzinski, G. Hess / Neuroscience Letters 298 (2001) 37±40
little GABA-ergic inhibitory transmission in contrast to super®cial layers of the motor cortex [5], recorded ®eld potentials are likely to re¯ect mostly monosynaptic excitatory synaptic activity [12]. Thus, this result may be interpreted as an effect of long-term, LTPK-related change on short-term, paired-pulse plasticity of excitatory synaptic transmission, perhaps mediated by the activation of protein kinase C [20]. Similar effect of a transient NMDA receptorindependent potentiation, induced by repetitive depolarizing pulses in the presence of APV, on paired-pulse facilitation has been observed using intracellular recordings from hippocampal pyramidal cells [7]. In conclusion, the present study demonstrates that intralaminar connections contained within layer V of adult rat motor cortex are readily modi®able in vitro by a VDCCsdependent mechanism. This mechanism may be potentially useful in forming new sensorimotor associations, however, its physiological signi®cance remains to be established. Supported by the Institute of Zoology grant BW/IZ/50/ 2000. G.H. is an International Research Scholar of the Howard Hughes Medical Institute. [1] Aniksztejn, L. and Ben-Ari, Y., Novel form of long-term potentiation produced by a K 1 channel blocker in the hippocampus, Nature, 349 (1991) 67±69. [2] Aroniadou, V.A. and Keller, A., The patterns and synaptic properties of horizontal intracortical connections in the rat motor cortex, J. Neurophysiol., 70 (1993) 1553±1569. [3] Aroniadou, V.A. and Keller, A., Mechanisms of LTP induction in rat motor cortex in vitro, Cereb. Cortex, 5 (1995) 353± 362. [4] Baranyi, A. and Feher, O., Long-term facilitation of excitatory synaptic transmission in single motor cortical neurones of the cat produced by repetitive pairing of synaptic potentials and action potentials following intracellular stimulation, Neurosci. Lett., 23 (1981) 303±308. [5] van Brederode, J.F.M. and Spain, W.J., Differences in inhibitory synaptic input between layer II-III and layer V neurons of the cat neocortex, J. Neurophysiol., 74 (1995) 1149±1166. [6] BuÈte®sch, C.M., Davis, B.C., Wise, S.P., Sawaki, L., Kopylev, L., Classen, J. and Cohen, L.G., Mechanisms of use-dependent plasticity in the human motor cortex, Proc. Natl. Acad. Sci. USA, 97 (2000) 3661±3665. [7] Chen, H.-X., Hanse, E., Pananceau, M. and Gustafsson, B.,
[8] [9] [10] [11]
[12]
[13]
[14]
[15] [16] [17] [18]
[19] [20]
Distinct expressions for synaptic potentiation induced by calcium through voltage-gated calcium and N-methyl-daspartate receptor channels in the hippocampal CA1 region, Neuroscience, 86 (1998) 415±422. Fox, K., The critical period for long-term potentiation in primary sensory cortex, Neuron, 15 (1995) 485±488. Grover, L. and Teyler, T.J., Two components of long-term potentiation induced by different patterns of afferent activation, Nature, 347 (1990) 477±479. Hanse, E. and Gustafsson, B., TEA elicits two distinct potentiations of synaptic transmission in the CA1 region of the hippocampal slice, J. Neurosci., 14 (1994) 5028±5034. Hess, G. and Donoghue, J.P., Long-term potentiation of horizontal connections provides a mechanism to reorganize cortical motor maps, J. Neurophysiol., 71 (1994) 2543±2547. Hess, G., Jacobs, K.M. and Donoghue, J.P., N-methyl-daspartate receptor-mediated component of ®eld potentials evoked in horizontal pathways of rat motor cortex, Neuroscience, 61 (1994) 225±235. Huang, Y.Y. and Malenka, R.C., Examination of TEAinduced synaptic enhancement in area CA1 of the hippocampus: the role of voltage-dependent Ca 21 channels in the induction of LTP, J. Neurosci., 13 (1993) 568±576. Huber, K.M., Mauk, M.D. and Kelly, P.T., Distinct LTP induction mechanisms: contribution of NMDA receptors and voltage-dependent calcium channels, J. Neurophysiol., 73 (1995) 270±279. Iriki, A., Pavlides, C., Keller, A. and Asanuma, H., Long-term potentiation in the motor cortex, Science, 245 (1989) 1385± 1387. Jacobs, K.M. and Donoghue, J.P., Reshaping the cortical motor map by unmasking latent intracortical connections, Science, 251 (1991) 944±947. Kimura, A., Caria, M.A., Melis, F. and Asanuma, H., Longterm potentiation within the cat motor cortex, NeuroReport, 5 (1994) 2372±2376. McCormick, D.A., Connors, B.W., Lighthall, J.W. and Prince, D.A., Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex, J. Neurophysiol., 54 (1985) 782±806. Pelletier, M.R. and Hablitz, J.J., Tetraethylammoniuminduced synaptic plasticity in rat neocortex, Cereb. Cortex, 6 (1996) 771±780. Ramakers, G.M.J., Pasinelli, P., van Beest, M., van der Slot, A., Gispen, W.H. and De Graan, P.N.E., Activation of preand postsynaptic protein kinase C during tetraethylammonium-induced long-term potentiation in the CA1 ®eld of the hippocampus, Neurosci. Lett., 286 (2000) 53±56.
www.elsevier.com/locate/neulet
Tetraethylammonium-induced long-term potentiation in layer V horizontal connections of rat motor cortex Przemyslaw Jagodzinski, Grzegorz Hess* Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Krakow, Poland Received 8 November 2000; received in revised form 27 November 2000; accepted 27 November 2000
Abstract The possibility for the induction of long-lasting synaptic plasticity by the potassium channel blocker tetraethylammonium (TEA) was investigated in adult rat motor cortex in vitro. Brief application of TEA (25 mM) resulted in a long-term potentiation (LTPK) of ®eld potentials evoked in layer V intralaminar connections by 59 ^ 17%. This effect could be prevented by preincubation with nifedipine, a voltage-dependent calcium channel blocker (20 mM), but not by Nmethyl-d-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV, 100 mM). LTPK induction resulted in a smaller relative potentiation of a response to the second pulse of a pair (60 ms interpulse interval). These results indicate a potential of layer V horizontal connections for a NMDA receptor-independent form of persistent synaptic enhancement. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Synaptic plasticity; Brain slice; K 1 channel; Ca 21 channel; Paired-pulse; Adult rat
Horizontally-oriented intracortical connections have been proposed to serve as a substrate for plastic reorganizations of adult motor cortex [16]. N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) of synaptic ef®cacy is a candidate mechanism of these reorganizations in mammalian species ranging from rats to humans [6,11]. In slice preparations of the hippocampus, where LTP mechanisms have been extensively investigated, apart from NMDA receptor-dependent LTP another form of LTP has been described which can be induced under conditions of NMDA receptor blockade by a very highfrequency afferent activation. This form of LTP is induced by the in¯ux of Ca 21 ions through voltage-dependent Ca 21 channels (VDCCs) of l-type [9]. Similarly, a transient exposure of hippocampal slices to a potassium channel blocker tetraethylammonium (TEA) results in a long-lasting increase of synaptic responses, referred to as LTPK, which depends on the Ca 21 in¯ux into post-synaptic cells through l-type VDCCs [1,13,20]. Neocortical VDCC-dependent forms of LTP have not been well explored. VDCCs are involved in LTP induction in layer II/III of the motor cortex of immature rats [3], however, since a potential for synaptic plasticity diminishes * Corresponding author. Tel.: 148-12-633-6377, ext. 2602; fax: 148-12-634-3716. E-mail address: [email protected] (G. Hess).
markedly with age, at least in the sensory cortices after the critical period [8], it is important to determine whether adult neocortex expresses this property. The induction of VDCCs-dependent LTPK has been reported for synaptic connections terminating on layer II/III pyramidal neurons of rat frontal cortex [19] but it is not known if this form of LTP is operable in layer V connections. To test this possibility we investigated the effects of TEA on synaptic transmission in layer V horizontal connections of the motor cortex (MI). Brain slices containing a part of MI were prepared from young adult female and male rats aged 6±9 weeks. Animals were deeply anesthetized with sodium pentobarbital applied intraperitoneally and decapitated. The brain was removed and immersed in chilled (7± 88C) arti®cial cerebrospinal ¯uid (ACSF) of the following composition (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3 and 15 d-glucose, bubbled with 95% O2-5% CO2. Parasagittal slices (450 mm) were cut at the distance of 2.0±3.0 mm from the brain midline using a vibrating microtome and transferred to a ¯uid-gas interface chamber perfused (1 ml/min) with warmed ACSF (34:5 ^ 0:58C). The humidi®ed atmosphere over the slices was saturated with a mixture of 95% O2±5% CO2. Concentric bipolar platinum/stainless steel stimulating electrodes were placed 1100±1300 mm below the cortical surface to activate ®bers running within layer V [12]. Constant-current or constant-voltage, 0.2 ms pulses were
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 0) 01 71 5- 8
38
P. Jagodzinski, G. Hess / Neuroscience Letters 298 (2001) 37±40
delivered at 0.033 Hz. Test stimulus intensity was adjusted to evoke a half-maximum synaptic response based on inputoutput curves. Field potentials were recorded using glass micropipettes ®lled with 0.25 M NaCl (3±6 MV) from sites located approx. 500 mm from the stimulation sites. Data were ampli®ed, ®ltered (0.1±500 Hz), acquired at a 10 kHz sampling rate and analyzed on- and off-line. The amplitude of the initial negative component of the response was used as a measure of the population excitatory synaptic current because this potential re¯ects monosynaptic current sink [2,12]. Intracellular recordings were obtained using sharp microelectrodes ®lled with 3 M K-acetate (60±80 MV). Only cells having a resting membrane potential greater than 270 mV, input resistance $20 MV and overshooting action potentials were recorded. Tetraethylammonium chloride, nifedipine and d,l-2-amino-5-phosphonovalerate (Sigma) were applied in the bathing ACSF. The results are presented as means ^ SEM unless noted. The exposure of slices to 25 mM TEA for 11 min resulted typically (nine of 11 slices) in a long-term increase of ®eld potentials (LTPK) evoked in layer V intralaminar connections. LTPK developed gradually over about 30 min after termination of TEA application and then remained stable for more than 1.5 h. In two slices responses returned to nearcontrol values. Fig. 1A shows that at this timepoint the average ®eld potential peak amplitude was increased to 159 ^ 17% of baseline (n 11). Layer V LTPK was not dependent on stimulus-evoked synaptic activity, since in the absence of testing electrical stimuli the long-term
response increase of comparable magnitude could be produced when tested 90 min after TEA application (not shown). Intracellular recordings from four layer V regularspiking neurons [18] demonstrated that TEA induced a similar increase of post-synaptic potentials (Fig. 1B). Among the set of neurons the post-synaptic potential peak amplitude increased by 22±64%. Consistent with previous studies [1,19] the effect was not due to permanently increased excitability of these cells (Fig. 1C). It has been reported that the hippocampal LTPK may consist of two components±one dependent on the activation of l-type calcium channels while the other, on NMDA receptors [10,14]. To test the involvement of both mechanisms in LTPK of the motor cortex in a next set of experiments TEA was applied in the presence of nifedipine, a calcium channel blocker. Pretreatment of the slices with 20 mM nifedipine did not markedly change recorded ®eld potentials, however, it blocked the long-term effect of TEA application. As illustrated in Fig. 2A, after a transient TEAinduced increase ®eld potential amplitude returned to 108 ^ 5% of control values (n 11) within 70 min. In contrast, the possibility for LTPK induction was not in¯uenced by d,l-2-amino-5-phosphonovalerate (APV, 100 mM), an antagonist of NMDA receptors. Among the four tested slices, in agreement with earlier ®ndings [12], the addition of APV to the ACSF resulted in a small reduction of late part of the ®eld potential beginning after the peak of the response. As shown in Fig. 2B, the application of TEA in the continuous presence of APV resulted in an increase of
Fig. 1. LTPK in layer V horizontal connections. (A1) Mean effect (^SEM, n 11) of 25 mM TEA applied for 11 min (bar) on intralaminar layer V ®eld potential amplitude. (A2) Superpositions of averages of ten responses recorded at times indicated in A1 during a representative experiment. Note a prolonged response timecourse at time 3, most likely due to enhanced polysynaptic transmission. (B1) LTPK of post-synaptic potentials recorded from a representative layer V neuron. Broken lines represent ^2 SD over baseline period. (B2) Superposition of averages (n 10) of post-synaptic potentials recorded as indicated in B1. (C) Response of the cell to a threshold, 400 ms long depolarizing current pulse applied before (control, 0.5 nA) and after (90 min, 0.6 nA) the termination of TEA application.
P. Jagodzinski, G. Hess / Neuroscience Letters 298 (2001) 37±40
39
Fig. 2. LTPK is blocked by nifedipine but not by APV. (A) Summary of experiments (^SEM, n 11) in which TEA was applied in the presence of nifedipine. Bars indicate the duration of drug applications. (B) Summary of experiments (^SEM, n 4) in which APV was present throughout the experiment and TEA was applied as indicated by a bar.
response to 152 ^ 8% of control values (n 4) 70 min after termination of TEA application. This result indicates that LTPK may be induced and expressed entirely by nonNMDA receptor-based synaptic transmission. To get further insight into LTPK mechanism, paired-pulse evoked ®eld potentials were analyzed. As demonstrated in Fig. 3, during the control period paired-pulse stimulation (60 ms interpulse interval) of layer V horizontal connections resulted in a slight facilitation of the response to the second pulse. On average its amplitude in relation to that evoked by the ®rst pulse (R2/R1) was 1:054 ^ 0:027 (n 8). However, after the induction of LTPK the R2/R1 ratio was reduced to 0:916 ^ 0:049 (P , 0:001, paired t-test), indicating a smaller degree of potentiation of the response to the second pulse. Our results demonstrate that the transient exposure of the slices containing a part of the motor cortex to 25 mM TEA results in a long-term increase of ®eld potentials evoked in layer V intralaminar connections. Earlier in vivo studies on adult motor cortical synaptic plasticity suggested that there may be layer differences in the potential for LTP. It has been noted that although some inputs to layer V pyramidal neurons may undergo activity-dependent long-lasting potentiation under speci®c experimental conditions [4,17], LTP is induced more readily in layers II±III [15]. The present results
complement the data provided by Pelletier and Hablitz [19] in showing that LTPK may be readily induced in deep layers of the rat neocortex. Similarly to that study and to the hippocampal LTPK [1], we note that layer V LTPK is characterized by a gradual increase of synaptic responses after TEA withdrawal. The mechanism of layer V LTPK induction is similar to that of layer II/III [19], since in both cases it is dependent on nifedipine-sensitive VDCCs, presumably of l-type, but not on NMDA receptors and does not require stimulationevoked synaptic activity. The involvement of NMDA receptors in LTPK induction in the hippocampus has been a matter of discussion. While the initial studies suggested that LTPK is entirely dependent on VDCCs [1,13], it was later demonstrated that it may have a NMDA receptor-dependent component [10] which required presynaptic stimulation [14]. The present results con®rm a lack of the involvement of NMDA receptors in neocortical LTPK [19], although it can not be excluded that NMDA receptors are involved in short-lasting effects of TEA application [14]. It has been reported that in the hippocampus LTPK does not interact with paired-pulse facilitation [13]. The present data, however, demonstrate a change in the relationship between responses evoked by two closely spaced pulses. Since the moderate electrical stimulation of layer V engages
Fig. 3. Interaction between LTPK and paired-pulse plasticity. (A) The paired-pulse ratio (amplitude of the second response/amplitude of the ®rst response, R2/R1) before (con) and after (post) LTPK induction (^SEM, n 8). (B) Superpositions of averages of ®eld potentials evoked by paired pulses before (thin line) and after (thick line) LTPK induction at 45 mA in a representative experiment (upper graph). Lower graph shows a superposition of above traces which were scaled to match the amplitude of the response to the ®rst pulse (R1). Note a difference in the amplitude of the second response (R2).
40
P. Jagodzinski, G. Hess / Neuroscience Letters 298 (2001) 37±40
little GABA-ergic inhibitory transmission in contrast to super®cial layers of the motor cortex [5], recorded ®eld potentials are likely to re¯ect mostly monosynaptic excitatory synaptic activity [12]. Thus, this result may be interpreted as an effect of long-term, LTPK-related change on short-term, paired-pulse plasticity of excitatory synaptic transmission, perhaps mediated by the activation of protein kinase C [20]. Similar effect of a transient NMDA receptorindependent potentiation, induced by repetitive depolarizing pulses in the presence of APV, on paired-pulse facilitation has been observed using intracellular recordings from hippocampal pyramidal cells [7]. In conclusion, the present study demonstrates that intralaminar connections contained within layer V of adult rat motor cortex are readily modi®able in vitro by a VDCCsdependent mechanism. This mechanism may be potentially useful in forming new sensorimotor associations, however, its physiological signi®cance remains to be established. Supported by the Institute of Zoology grant BW/IZ/50/ 2000. G.H. is an International Research Scholar of the Howard Hughes Medical Institute. [1] Aniksztejn, L. and Ben-Ari, Y., Novel form of long-term potentiation produced by a K 1 channel blocker in the hippocampus, Nature, 349 (1991) 67±69. [2] Aroniadou, V.A. and Keller, A., The patterns and synaptic properties of horizontal intracortical connections in the rat motor cortex, J. Neurophysiol., 70 (1993) 1553±1569. [3] Aroniadou, V.A. and Keller, A., Mechanisms of LTP induction in rat motor cortex in vitro, Cereb. Cortex, 5 (1995) 353± 362. [4] Baranyi, A. and Feher, O., Long-term facilitation of excitatory synaptic transmission in single motor cortical neurones of the cat produced by repetitive pairing of synaptic potentials and action potentials following intracellular stimulation, Neurosci. Lett., 23 (1981) 303±308. [5] van Brederode, J.F.M. and Spain, W.J., Differences in inhibitory synaptic input between layer II-III and layer V neurons of the cat neocortex, J. Neurophysiol., 74 (1995) 1149±1166. [6] BuÈte®sch, C.M., Davis, B.C., Wise, S.P., Sawaki, L., Kopylev, L., Classen, J. and Cohen, L.G., Mechanisms of use-dependent plasticity in the human motor cortex, Proc. Natl. Acad. Sci. USA, 97 (2000) 3661±3665. [7] Chen, H.-X., Hanse, E., Pananceau, M. and Gustafsson, B.,
[8] [9] [10] [11]
[12]
[13]
[14]
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