Neuroscience Letters, 141 (1992) 262-264 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
262
NSL 08776
Development of long-term potentiation in the somatosensory cortex of rats of different ages I. Vil~igi a, I. T a r n a w a b a n d I. Banczerowski-Pelyhe a "Department qf Comparative Physiology, E6tvOs Lor6nd University, Budapest (Hungary) and blnstitutefor Drug Research, Budapest (Hungary) (Received 5 March 1992; Revised version received 14 April 1992; Accepted 24 April 1992)
Key words'." Long-term potentiation; Somatosensory cortex; Ontogeny; Rat; In vitro slice The age dependence of possible long-term potentiation (LTP) induction in rat somatosensory cortex was studied in in vitro slice experiments. Coronal slices were prepared from the somatosensory cortex of rats of different ages, and excitatory postsynaptic potentials evoked by stimulation of the white matter (0.1 Hz, subthreshold for spike) were recorded intracellularly. In 70% of the slices taken from 2-week-old rats, a moderate potentiation (20-30%) could be induced by either 5 or 100 Hz stimulation. No LTP was observed in younger (1 week) or older (3 weeks) cortex. On the basis of our experiments an important ontogenetic role of increased synaptic efficacy is suggested in a critical developmental period of rats after birth.
Brief tetanic stimulation of afferent fibres may trigger some sustained biochemical processes in the hippocampus and in other cortical regions resulting in a long-lasting increase in synaptic strength or efficacy. The phenomenon of long-term potentiation (LTP) can be studied in different in vivo and in vitro experimental models [1,3, 7, 18]. Activation of glutamate receptor-mediated processes, first of all the N M D A types, has been shown to be involved in the formation of LTP [15]. The blockade of LTP has been well demonstrated with the aid of different glutamate receptor antagonists [6]. A search for the role of non-NMDA receptor-mediated processes has been in progress, since strong and specific non-NMDA antagonists have been synthesized [8]. LTP is regarded as a model of learning processes at the cellular level, in the hippocampus [4, 6, 23] or in other cortical regions [12, 20], although there are also some arguments against the relevance of the model [13]. The LTP development depends on several factors, such as the brain area or the age of the animals, etc. [5, 19, 20]. In adult rats, for example, LTP can easily be evoked in the hippocampus. In the neocortex, however, it usually develops only in the presence of a low concentration of the GABAA antagonist bicucullin [2]. Correspondence: I. Vil~igi, Department of Comparative Physiology, E6tv6s Lor~ind University, 1088, Budapest, Mtazeum krt. 4/a, Hungary.
Different investigators use a wide range of stimulus frequencies (from 2 to 400 Hz) [2, 16]. In some cases, the tetanic stimuli elicit not a potentiation but rather a depression depending on the frequency and also on the strength of the stimulation [2]. Lower stimulation frequency and strength frequently evoke long-term depression in spite of the potentiation [11, 16, 22, 23]. Although studies of LTP in developing animals are relatively few, it has become clear that there is an age period after birth when LTP can be evoked more easily in all brain areas studied so far [12, 14, 16, 20, 23]. In our earlier in vitro experiments on rat cortical slices we demonstrated that there is a critical period between the 2nd and the 3rd week after birth when seizure activity develops very easily [24]. The development and maintenance of the seizure activity depends on the N M D A and AMPA receptor-mediated processes and these receptors play an important role in the control of some ontogenetic events [8]. The N M D A receptors play an important role not only in the seizure and ontogenic processes but also in the development of the LTP. The goal of our study was to investigate the possibility of the development of LTP in rat somatosensory cortex slices in this sensitive period and to compare the data to those obtained in the seizure-susceptibility experiments. In these series of experiments coronal slices were prepared from the somatosensory cortex of rats of different ages (from 7- to 22-day-old rats were selected into 3 age
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Fig. 1. Formation of LTP after 5 Hz tetanic stimulation. A: the evoked responses with 0.1 Hz test-stimulation, gradually increasing the stimulation voltage from the EPSP-threshold to the spike-threshold, before the tetanic stimulation and alter it. The bottom part of the figure shows recordings with a lower paper speed, the top part shows those with a faster paper speed. Horizontal calibration: 100 s (bottom part) or 50 ms (top part). B: the stimulus intensity response amplitude curve. Responses with different stimulation strengths, t : bet\~re the tetanic stimulation; ~ : after the tetanic stimulation. The potentiation is stronger at the higher stimulation voltages, and the EPSP-threshold is decreased after tetanus.
groups: 1-week-old (7 10 days), 2-week-old (13 16 days) and 3-week-old (19 22 days). Under ether narcosis, the brain was quickly removed into an ice-cold incubation solution, and 400-#m-thick slices were cut with a vibratome. During the experiments we used an interface type slice chamber, artificial perfusion solution (composition in raM: 126 NaC1, 1.8 KC1, 1.25 KH2POa, 1.3 MgSOa, 26 NaHCO~, 2.4 CaCI> 10 glucose), 95% 02/5% CO2 bubbling, 34.5_+0.5°C temperature. The intracellular recording electrode was filled with 3 tool KC1 and cells were impaled in the layer IIl. The bipolar platinum stimulating electrode was positioned at the border of the white and grey matter below the recording electrode. In the cells that were selected for the LTP experiment, stimulation evoked excitatory postsynaptic potentials (EPSPs), the peak amplitude of which was basically determined by a short latency (presumably monosynaptic) component. The frequency of the tetanic stimulation was either 5 Hz (for 1 min) or 100 Hz (for 4 times 5 s/l min), with an intensity subthreshold for spike. In one half of the experiments the intensity of the test stimulation was gradually increased in 0.1 V steps with 10 s intervals starting from the EPSP threshold up to the spike threshold, and the evoked responses were recorded before the tetanic stimulation and 5, 10 and 20 rain after it. In the other half of the experiments continuous stimulation at 0.1 Hz frequency was applied with a fixed intensity, and the changes of the evoked responses were recorded for more than 20 rain afte," the tetanic stimulation. Fhe criterion of the LTP was defined as at least 20% increase in peak amplitude 20 min after the tetanus. There was no essential difference in respect of the membrane potential (MP), the input resistance (R) at 0.2 nA values and the threshold of the EPSPs and spikes
among the 3 age groups (Table I). Most of the cells investigated belonged to the regular firing type of neurons [9]. Action potentials were. usually followed by a large afterhyperpolarization. It can be supposed that the membrane parameters had already developed at the age when the investigations started (6 days postnatally), although the biochemical differentiation was still in progress. In the case of the 2-week-old rats we were able to evoke LTP in 5 of the 6 slices tested when 5 Hz was used and in 3 of the 6 slices with 100 Hz tetanic stimulation. The peak amplitude of the EPSP was 132.9_+29.1% of the control value in the first and 119.4+14.2% in the latter TABLE I The resting membrane potential (MP), the input resistance (&) at a 0.2 nA conductance-pulse, the EPSP-threshold and the spike-threshold can be seen in the table. In the I- and 3-week-old rats we could not evoke LTP at all. It was only possible in the 2-week-old rats with both 5 and 100 Hz tetanic stimulation frequency. The 5 Hz stimulation frequency proved to be more effective. MP (mV)
1 week (7 10 days) n-6
R~ (MD)
LTP StimuSpike lation threshold 5 Hz 100 Hz threshold
69.75+5.7 51.3+21.0 3.8+1.8
4.4! 1.6
0/3
0/3
75.3+_3.7 2 weeks (13 16 days) n-12
39.2+10.9 3.1+_0.75
t.5rl.28
5/6
3/6
75.8+_1.7 3 weeks (19 22 days) n 8
53.1+17.5 2.5+1.2
3.7+2.1
0/4
0/4
264
case. Not only was the amplitude of each response increased after tetanization, but lower stimulation intensity was enough to evoke EPSP in all slices where the stimulus intensity-response amplitude curve was determined. In those experiments where the test stimuli were gradually increased from the EPSP threshold up to the spike threshold, a near-parallel shift in the stimulus intensity response amplitude curve was seen with a slightly bigger potentiation at higher stimulation intensities (Fig. 1). It is not probable that a decrease of the threshold of the afferent fibers is responsible for the altered threshold for the EPSP. An increased synaptic efficacy, in which both pre- and postsynaptic events are involved, may explain the shift of the curve. We were not able to evoke LTP at all in the slices prepared either from 1- or from 3-week-old rats (Table I). Thus, our results suggest the existence of a sensitive period at around the 2nd week after birth, when the synaptic efficacy can be enhanced relatively easily. Our results are in accordance with those obtained in other brain regions or in other species [20, 16]. The exact time of the appearance of this sensitive period may depend on the brain region and the species used. This may be a very important period during the development of the brain, because the specific connections of basic neuronal networks form at this time: namely, the thalamic afferents grow into the somatosensory cortex and form their specific connections. The GABAergic inhibition has not fully developed yet at this early ontogenic period [17], so the predominance of excitatory processes can be observed. This condition may favour the development of synaptic potentiation. We suppose that the higher synaptic efficacy during the critical period may play a crucial role also in the developmental processes. LTP-like physiological processes, which are mainly regarded as important in learning and memory, may also play a role in developmental synaptic plasticity at an earlier ontogenic stage. This work was supported by a Grant from the Hungarian Government (OTKA I/3: 624). 1 Alkon, D.L., Amaral, D.G., Bear, M.F. et al., Learning and memory, Brain Res. Rev., 16 (1991) 193--220. 2 Artola, A., Br6cher, S. and Singer, W., Different voltage-dependent thresholds for inducing long-term depression and long-term potentiation in slices of rat visual cortex, Nature, 347 (1990) 69-72. 3 Baranyi, A. and Szente, M.B., Long-lasting potentiation ofsynaptic transmission requires postsynaptic modifications in the neocortex, Brain Res., 423 (1987) 378-384. 4 Bashir, Z.I., Alford, S., Davies, S.N. et al., Long-term potentiation of NMDA receptor-mediated synaptic transmission in the hippocampus, Nature, 349 (1991) 156- 158. 5 Baskys, A., Reynolds, J.N. and Carlen, P.L.. NMDA depolariza-
tions and long-term potentiation are reduced m tfic aged ~at n~
262
NSL 08776
Development of long-term potentiation in the somatosensory cortex of rats of different ages I. Vil~igi a, I. T a r n a w a b a n d I. Banczerowski-Pelyhe a "Department qf Comparative Physiology, E6tvOs Lor6nd University, Budapest (Hungary) and blnstitutefor Drug Research, Budapest (Hungary) (Received 5 March 1992; Revised version received 14 April 1992; Accepted 24 April 1992)
Key words'." Long-term potentiation; Somatosensory cortex; Ontogeny; Rat; In vitro slice The age dependence of possible long-term potentiation (LTP) induction in rat somatosensory cortex was studied in in vitro slice experiments. Coronal slices were prepared from the somatosensory cortex of rats of different ages, and excitatory postsynaptic potentials evoked by stimulation of the white matter (0.1 Hz, subthreshold for spike) were recorded intracellularly. In 70% of the slices taken from 2-week-old rats, a moderate potentiation (20-30%) could be induced by either 5 or 100 Hz stimulation. No LTP was observed in younger (1 week) or older (3 weeks) cortex. On the basis of our experiments an important ontogenetic role of increased synaptic efficacy is suggested in a critical developmental period of rats after birth.
Brief tetanic stimulation of afferent fibres may trigger some sustained biochemical processes in the hippocampus and in other cortical regions resulting in a long-lasting increase in synaptic strength or efficacy. The phenomenon of long-term potentiation (LTP) can be studied in different in vivo and in vitro experimental models [1,3, 7, 18]. Activation of glutamate receptor-mediated processes, first of all the N M D A types, has been shown to be involved in the formation of LTP [15]. The blockade of LTP has been well demonstrated with the aid of different glutamate receptor antagonists [6]. A search for the role of non-NMDA receptor-mediated processes has been in progress, since strong and specific non-NMDA antagonists have been synthesized [8]. LTP is regarded as a model of learning processes at the cellular level, in the hippocampus [4, 6, 23] or in other cortical regions [12, 20], although there are also some arguments against the relevance of the model [13]. The LTP development depends on several factors, such as the brain area or the age of the animals, etc. [5, 19, 20]. In adult rats, for example, LTP can easily be evoked in the hippocampus. In the neocortex, however, it usually develops only in the presence of a low concentration of the GABAA antagonist bicucullin [2]. Correspondence: I. Vil~igi, Department of Comparative Physiology, E6tv6s Lor~ind University, 1088, Budapest, Mtazeum krt. 4/a, Hungary.
Different investigators use a wide range of stimulus frequencies (from 2 to 400 Hz) [2, 16]. In some cases, the tetanic stimuli elicit not a potentiation but rather a depression depending on the frequency and also on the strength of the stimulation [2]. Lower stimulation frequency and strength frequently evoke long-term depression in spite of the potentiation [11, 16, 22, 23]. Although studies of LTP in developing animals are relatively few, it has become clear that there is an age period after birth when LTP can be evoked more easily in all brain areas studied so far [12, 14, 16, 20, 23]. In our earlier in vitro experiments on rat cortical slices we demonstrated that there is a critical period between the 2nd and the 3rd week after birth when seizure activity develops very easily [24]. The development and maintenance of the seizure activity depends on the N M D A and AMPA receptor-mediated processes and these receptors play an important role in the control of some ontogenetic events [8]. The N M D A receptors play an important role not only in the seizure and ontogenic processes but also in the development of the LTP. The goal of our study was to investigate the possibility of the development of LTP in rat somatosensory cortex slices in this sensitive period and to compare the data to those obtained in the seizure-susceptibility experiments. In these series of experiments coronal slices were prepared from the somatosensory cortex of rats of different ages (from 7- to 22-day-old rats were selected into 3 age
263
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........
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STIMULUS
2'7
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INTENSITY
al,
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Fig. 1. Formation of LTP after 5 Hz tetanic stimulation. A: the evoked responses with 0.1 Hz test-stimulation, gradually increasing the stimulation voltage from the EPSP-threshold to the spike-threshold, before the tetanic stimulation and alter it. The bottom part of the figure shows recordings with a lower paper speed, the top part shows those with a faster paper speed. Horizontal calibration: 100 s (bottom part) or 50 ms (top part). B: the stimulus intensity response amplitude curve. Responses with different stimulation strengths, t : bet\~re the tetanic stimulation; ~ : after the tetanic stimulation. The potentiation is stronger at the higher stimulation voltages, and the EPSP-threshold is decreased after tetanus.
groups: 1-week-old (7 10 days), 2-week-old (13 16 days) and 3-week-old (19 22 days). Under ether narcosis, the brain was quickly removed into an ice-cold incubation solution, and 400-#m-thick slices were cut with a vibratome. During the experiments we used an interface type slice chamber, artificial perfusion solution (composition in raM: 126 NaC1, 1.8 KC1, 1.25 KH2POa, 1.3 MgSOa, 26 NaHCO~, 2.4 CaCI> 10 glucose), 95% 02/5% CO2 bubbling, 34.5_+0.5°C temperature. The intracellular recording electrode was filled with 3 tool KC1 and cells were impaled in the layer IIl. The bipolar platinum stimulating electrode was positioned at the border of the white and grey matter below the recording electrode. In the cells that were selected for the LTP experiment, stimulation evoked excitatory postsynaptic potentials (EPSPs), the peak amplitude of which was basically determined by a short latency (presumably monosynaptic) component. The frequency of the tetanic stimulation was either 5 Hz (for 1 min) or 100 Hz (for 4 times 5 s/l min), with an intensity subthreshold for spike. In one half of the experiments the intensity of the test stimulation was gradually increased in 0.1 V steps with 10 s intervals starting from the EPSP threshold up to the spike threshold, and the evoked responses were recorded before the tetanic stimulation and 5, 10 and 20 rain after it. In the other half of the experiments continuous stimulation at 0.1 Hz frequency was applied with a fixed intensity, and the changes of the evoked responses were recorded for more than 20 rain afte," the tetanic stimulation. Fhe criterion of the LTP was defined as at least 20% increase in peak amplitude 20 min after the tetanus. There was no essential difference in respect of the membrane potential (MP), the input resistance (R) at 0.2 nA values and the threshold of the EPSPs and spikes
among the 3 age groups (Table I). Most of the cells investigated belonged to the regular firing type of neurons [9]. Action potentials were. usually followed by a large afterhyperpolarization. It can be supposed that the membrane parameters had already developed at the age when the investigations started (6 days postnatally), although the biochemical differentiation was still in progress. In the case of the 2-week-old rats we were able to evoke LTP in 5 of the 6 slices tested when 5 Hz was used and in 3 of the 6 slices with 100 Hz tetanic stimulation. The peak amplitude of the EPSP was 132.9_+29.1% of the control value in the first and 119.4+14.2% in the latter TABLE I The resting membrane potential (MP), the input resistance (&) at a 0.2 nA conductance-pulse, the EPSP-threshold and the spike-threshold can be seen in the table. In the I- and 3-week-old rats we could not evoke LTP at all. It was only possible in the 2-week-old rats with both 5 and 100 Hz tetanic stimulation frequency. The 5 Hz stimulation frequency proved to be more effective. MP (mV)
1 week (7 10 days) n-6
R~ (MD)
LTP StimuSpike lation threshold 5 Hz 100 Hz threshold
69.75+5.7 51.3+21.0 3.8+1.8
4.4! 1.6
0/3
0/3
75.3+_3.7 2 weeks (13 16 days) n-12
39.2+10.9 3.1+_0.75
t.5rl.28
5/6
3/6
75.8+_1.7 3 weeks (19 22 days) n 8
53.1+17.5 2.5+1.2
3.7+2.1
0/4
0/4
264
case. Not only was the amplitude of each response increased after tetanization, but lower stimulation intensity was enough to evoke EPSP in all slices where the stimulus intensity-response amplitude curve was determined. In those experiments where the test stimuli were gradually increased from the EPSP threshold up to the spike threshold, a near-parallel shift in the stimulus intensity response amplitude curve was seen with a slightly bigger potentiation at higher stimulation intensities (Fig. 1). It is not probable that a decrease of the threshold of the afferent fibers is responsible for the altered threshold for the EPSP. An increased synaptic efficacy, in which both pre- and postsynaptic events are involved, may explain the shift of the curve. We were not able to evoke LTP at all in the slices prepared either from 1- or from 3-week-old rats (Table I). Thus, our results suggest the existence of a sensitive period at around the 2nd week after birth, when the synaptic efficacy can be enhanced relatively easily. Our results are in accordance with those obtained in other brain regions or in other species [20, 16]. The exact time of the appearance of this sensitive period may depend on the brain region and the species used. This may be a very important period during the development of the brain, because the specific connections of basic neuronal networks form at this time: namely, the thalamic afferents grow into the somatosensory cortex and form their specific connections. The GABAergic inhibition has not fully developed yet at this early ontogenic period [17], so the predominance of excitatory processes can be observed. This condition may favour the development of synaptic potentiation. We suppose that the higher synaptic efficacy during the critical period may play a crucial role also in the developmental processes. LTP-like physiological processes, which are mainly regarded as important in learning and memory, may also play a role in developmental synaptic plasticity at an earlier ontogenic stage. This work was supported by a Grant from the Hungarian Government (OTKA I/3: 624). 1 Alkon, D.L., Amaral, D.G., Bear, M.F. et al., Learning and memory, Brain Res. Rev., 16 (1991) 193--220. 2 Artola, A., Br6cher, S. and Singer, W., Different voltage-dependent thresholds for inducing long-term depression and long-term potentiation in slices of rat visual cortex, Nature, 347 (1990) 69-72. 3 Baranyi, A. and Szente, M.B., Long-lasting potentiation ofsynaptic transmission requires postsynaptic modifications in the neocortex, Brain Res., 423 (1987) 378-384. 4 Bashir, Z.I., Alford, S., Davies, S.N. et al., Long-term potentiation of NMDA receptor-mediated synaptic transmission in the hippocampus, Nature, 349 (1991) 156- 158. 5 Baskys, A., Reynolds, J.N. and Carlen, P.L.. NMDA depolariza-
tions and long-term potentiation are reduced m tfic aged ~at n~
