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Partial hippocampal kindling increases paired-pulse facilitation and burst frequency in hippocampal CA1 neurons
Neuroscience Letters, 154 (1993) 191 194 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
191
NSL 09520
Partial hippocampal kindling increases paired-pulse facilitation and burst frequency in hippocampal CA1 neurons Dichen Zhao and L. Stan Leung Departments of Clinical Neurological Sciences and Physiology, University of Western Ontario, London, Ontario, Canada (Received 4 December 1992; Revised version received 22 February 1993; Accepted 22 February 1993)
Key words: Kindling; Intracellular recording; Afterhyperpolarization; Membrane property; Presynaptic facilitation For up to 3 weeks after 15 evoked afterdischarges (partial kindling) in the hippocampus in vivo, paired-pulse facilitation of the CAI apical dendritic excitatory postsynaptic potentials (EPSPs), recorded from single neurons in vitro, was significantly larger in neurons of kindled than control rats. Partial kindling did not significantly affect the resting membrane potential, the threshold or size of the action potential (AP), the fast afterhyperpolarization, input resistance, time constant or the EPSP threshold. The number of APs induced within the initial 20 ms of a long-duration 0.5-nA depolarizing current was significantly higher in the kindled than control neurons. The increase in paired-pulse facilitation and intrinsic spiking in hippocampal CA1 after partial kindling may contribute to an increase in seizure susceptibility.
Seizures are known to cause long-lasting changes. Febrile seizure may induce complex partial seizures in adults [16]. Experimentally induced afterdischarges in the brain, if delivered repetitively (the kindling paradigrn) also induced persistent seizure susceptibility [3, 12]. The underlying cause of the kindling-induced seizure susceptibility is not known. In the hippocampus, it may be caused by an increase in the excitatory postsynaptic potentials (EPSPs) [1, 4, 9, 12, 13, 21], increased Ca 2÷ influx [18], loss of inhibition [4-6, 17, 21, 22] or increased intrinsic neuronal bursting [6, 12, 20]. The present study is part of an on-going effort to understand the physiological changes induced by seizures, using kindling as the experimental model. The rationale was to test the hypothesis that kindling induced an increase in the paired-pulse facilitation of the EPSPs in single cells of CA1 in vitro, as suggested by previous studies on the population EPSPs in CA1 [4, 6, 21]. The kindling was partial, i.e., delivered to a stage before generalized convulsions, to reveal the mechanisms involved in the early stage of epileptogenesis. The surgical, kindling and in vitro slice preparation procedures have been reported elsewhere [8, 9]. Briefly, kindling was delivered through chronic-indwelling electrodes implanted in stratum moleculare of hippocampal Correspondence. L.S. Leung, Department of Physiology, Health Sciences, University of Western Ontario, Medical Sciences Building, London, Ontario N6A 5A5, Canada. Fax: (1) (519) 6613827.
CA1 of male Long Evans rats. Experimental rats were given the kindling stimulus (1 s, 100 Hz, 0.1-ms square pulses) while control rats were given low-frequency stimulations (LFSs), each consisting of 100 pulses at 0.167 Hz. 15 ADs/LFSs were delivered hourly, 5/day over 3 days. On days 1-3 (day-1 group) or 21-23 (day-23 group) after the last day of kindling or control treatment, 500¢tm-thick slices were obtained from the middle part of the stimulated hippocampus of each rat [10, 21]. In some experiments, hippocampal slices were obtained from Wistar rats which were not chronically implanted with electrodes (the unstimulated control group). The in vitro slice was perfused with artificial cerebrospinal fluid of the after composition (mM): NaCI, 124; KC1, 5; NaH2PO4.H20, 1.25; MgSO4"7H20, 2; CaC12" 6H20, 2; NaHCO3, 26; and glucose, 10. A concentric bipolar stimulating electrode was placed at the Schaffer collaterals (stratum radiatum in CA1, on the CA3 side). CA1 neurons were recorded at the cell body layer [resting membrane potential of < -60 mV and overshooting action potentials (APs)] using micropipettes filled with 3 M potassium acetate. The potentials were amplified by an Axon-IA probe, digitized (at 10 KHz) and stored on line by a custom program [10]. A holding hyperpolarization current (< 0.19 nA) was sometimes necessary to suppress spontaneous firing. Input resistance (Rin), time constant, spike height were measured as described elsewhere [10]. The fast afterhyperpolarization (AHP) was measured at 1-5 ms after the spike peak. Paired-pulse stimulations
192
A
were given to the Schaffer collaterals at 0.1 Hz and interpulse intervals (IPIs) of 10- 200 ms. At least two stimulus intensities were used, one just below and one just above the spike threshold of the first pulse• The slope of the EPSP was measured over a 1.5-ms interval near the onset of the EPSP, which was typically at 1.5-2 ms from the shock artifact. The points used for the EPSP measure were clearly before the onset of the spike, for the first (El) or second pulse (E2). ANOVA and non-parametric (Wilcoxon) tests were used for testing statistical significance, using the number of neurons as the independent variable. All kindled rats remained at seizure stage I after 15 ADs, which was expected from a previous study [8]. 72 neurons were impaled in slices derived from 22 rats (Table I). There was no significant difference between neurons from kindled and control rats in the resting membrane potential, input resistance, time constant, the height, voltage and current threshold of the AP and fast AHP (Table I). The electrophysiological characteristics were typical of CAI pyramidal cells [10]. When injected with a long-duration depolarizing current, the typical firing pattern of CA1 neurons was a series of spikes that gradually decreased their interspike intervals (Fig. 1A). In some cells, however, a clustering of the early spikes (bursting) superimposed on a slow depolarizing hump was observed (Fig. IB). Some bursting cells were found in kindled and control groups and the occurrence of bursting neurons was only statistically different (P < 0.05, Z 2) in kindled than in control slices for day 1 but not for day 23. Except for a lower spike threshold current in bursting than non-bursting neurons, the membrane properties listed in Table I were not significantly different between the eight bursting and seven non-bursting neurons in the day-1 kindled group. However, we found it difficult to categorize bursting in some
-~
75ms~1
B
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daylc - daylk
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day23c
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"~
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~ ~
.5-
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g0
~,.~.~ -.~,=.~_~
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8b ld0 1~0 110 td0
l a t e n c y a f t e r c u r r e n t injection (ms)
Fig. 1. Kindling induced changes in neuronal firing pattern. A,B: Examples of firing patterns induced by 2-3 times the threshold intensity of a long-duration (170-210 ms) depolarization current. A: A non-bursting neuron from a kindled day-23 slice; B: A bursting neurons from a kindled day-I rat. C: AP number/20-ms interval at various latencies after a 0.5-nA depolarization current.
CA1 neurons since the clustering of spikes and the slow hump may not be as clear as in Fig. 1. Thus, we quantified the spike frequency over short periods of time (20 ms) after the onset of a 0.5-nA depolarizing current. As expected, the spike frequency declined with time (accommodated) after the current onset [7]. The firing frequency for the initial 20 ms was significantly higher in the kindled than control neurons (Fig. 1C; Wilcoxon, P < 0.05,
TABLE I M E M B R A N E P R O P E R T I E S O F CAI P Y R A M I D A L CELLS F R O M K I N D L E D A N D C O N T R O L RATS Day 1
Day 23
Kindled
Control
Kindled
Control
Non-stimulated
N u m b e r of rats N umber of cells Resting potential (mV) Input resistance (MI2) Time constant (ms) Spike height(mV) Spike threshold (nA) Spike threshold (mV) A H P (mV)
8 15 71.7 47.1 11.1 72.0 0.20 12.67 2.5
6 18 70.2 48.8 12.4 70.6 0.29 14.87 2.57
4 16 72.9 43.0 10.5 77.5 0.18 11.1 2.15
4 12 74.5 45.2 11.9 71.4 0.26 15.32 2.4
Percent of bursting cells (%)
53.5
7 11 69.8 44.5 12.7 67.6 0.24 15.1 1.35 27.3
+_ 1.9 + 2.4 _+0.7 -+2.2 _+ 0.06 _+ 2.31 -+ 0.67
22.2
+_ 1.37 _+ 3.11 +0.7 + 1.43 _+ 0.05 -+ 1.42 -+ 0.61
12.5
+ 1.1 + 2.8 -+0.9 _+ 1.6 _+ 0.05 + 0.71 _+ 0.44
16.7
+ 1.6 _+ 2.7 -+1.2 _+ 2.6 + 0.07 + 1.97 +_ 0.45
_+ 2.5 _+ 6.9 _+ 1.4 +_ 3.0 + 0.06 +_ 1.9 _+ 0.12
193 for day 1 or 23 kindled vs. their respective controls) but not significantly different between different (day-1 or day-23) kindled groups or among different control groups. The stimulus threshold for evoking an AP in CA 1 neurons (< 30 I2A and < 0.15 ms duration) was not significantly different between control and kindled neurons. The synaptic responses evoked by stimuli at just above and just below the spike threshold were practically the same, whether a first spike (FS) or no FS was evoked (Fig. 2). The ratio of the slope of the EPSP evoked by E2 to that evoked by El, E2/E1, was significantly larger in kindled than control neurons, on either day 1 or 23 (Fig. 2). In a two-factor (experimental group x IPI) repeated measure ANOVA, there was a significant difference in E2/EI between kindled and control groups (P < 0.01 for all F evaluated between kindled and control groups for the four conditions: no FS-day 1, yes FS-day 1, no FSday 23 and yes FS-day 23t. There was no significant interaction between IPI and experimental group. E2/E1 was not different between yes FS and no FS groups and E1 was not significantly different among kindled and control groups. Previous studies reported that no changes were observed in the resting membrane potential, input resistance, spike amplitude and time constant of CA1 neurons after full kindling (generalized convulsions) of the commissure or perforant path [15, 20]. We confirmed the latter result using partial kindling. In addition, we observed a significant increase in the intrinsic excitability of CA1 neurons as tested by a depolarizing current. This was somewhat similar to the observation of a large number of bursting cells in 'kindled' slices by Yamada and Bilkey [20], though we, and others, also observed a large number of bursting CA 1 cells in control (low-frequency stimulated or unstimulated) slices [10, 11, 19]. We confirmed that kindling induced a significant increase in paired-pulse facilitation of the intracellularly recorded EPSPs. This facilitation was equally strong on days 1 and 23 after kindling (Fig. 2) and did not depend on whether the first pulse evoked a spike or not, i.e., E2 was minimally dependent on the A H P at - I 0 200 ms after the spike (which could reach several mV in amplitude). Paired-pulse EPSP facilitation may be caused by a presynaptic facilitation [2, 14] or a loss of postsynaptic (feedforward or feedback) inhibition. Our preliminary analysis of the neuronal hyperpolarization after low intensity afferent stimulation revealed no statistical difference between kindled and control groups but this presumed measure of the inhibition is tempered by other events (e.g., late EPSPs). The intracellular measure of the EPSP facilitation was found to be robust. The variance of intracellular E2/E1
FS
A 1.8-
1.4-
.C
-50
6
5'o
16o
1go
260
2~o
•.o- k-noFS v. k-FS -6-c-noFS --~ c-FS
B 1.8-
1.4-
.G
-s0
6
5'0
t60
~o
260
2~0
IPI (ms) Fig. 2. Inset on right top: Overlaid pair-pulse responses at a stimulus intensity that slightly exceeded the threshold of the FS and slightly below the FS threshold (no FS). IPI = 40 ms, from a control slice. Note the EPSP slopes and latency of the spike evoked by the second pulse were not affected by the presence or absence o f F S . E2/EI as a function of the IPI was plotted for day-I (A) and day-23 groups (B). In each graph, there are four subgroups: kindled no FS (k-noFS), kindled FS (k-FS), control no FS (c-noFS) and control FS (c-FS). For clarity, error bars for the four graphs are not included. N u m b e r of neurons (kindled no FS, kindled FS, control no FS and control FS): 12,10, 13,14 (A); 15,16, 17,17 (B).
was typically smaller than that of the extracellular (population) EPSPs [21], though the means of the extracelluar and intracellular EPSP facilitation was about the same. The use of low stimulus intensities in this study (~1.2 times the EPSP threshold) was made possible by the large signal to noise ratio of intracellular recordings. The kindling-induced changes reported here will tend to make CA1 more excitable and may contribute to seizure susceptibility. EPSP facilitation was shown to sum up during repeated pulses and may be an important factor in the spread and synchronization of large areas of
194
the b r a i n d u r i n g seizures. T h u s , s h o r t - t e r m plasticity, in a d d i t i o n to l o n g - t e r m p o t e n t i a t i o n [1, 12], s h o u l d be furt h e r s t u d i e d as a m e c h a n i s m a n d h u m a n seizures.
underlying experimental
11
12 We t h a n k B. S h e n for t e c h n i c a l assistance. T h e research was supported by NS-25383, NSERC H o s p i t a l for Sick C h i l d r e n F o u n d a t i o n 91 --074.
A1037 and
(Toronto) XG
l Cain, D.P., Long-term potentiation and kindling: how similar are the mechanisms? Trends Neurosci., 12 (1989) ~10. 2 Foster, T.C. and McNaughton, B.L., Long-term enhancement of CAI synaptic transmission is due to increased quantal size, not quantal content, Hippocampus, 1 (1991) 79 92. 3 Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295 330. 4 Kamphuis, W., Lopes da Silva, F.H. and Wadman, W.J., Changes in local evoked potentials in the rat hippocampus (CA1) during kindling epileptogenesis, Brain Res., 440 (1988) 205--2t 5. 5 Kamphuis, W., Gorter, J.A. and Lopes da Silva, F., A long-lasting decrease in the inhibitory effect of GABA on glutamate responses of hippocampal pyramidal neurons induced by kindling epileptogenesis, Neuroscience, 41 ( 1991) 425-431. 6 King, G.L., Dingledine, R., Giacchino, J.L. and McNamara, J.O,, Abnormal neuronal excitability in hippocampal slices from kindled rats, J. Neurophysiol., 54 (1985) 1295 1304. 7 Lanthorn, T., Storm, J. and Andersen, R, Current-to-frequency transduction in CAI hippocampal pyramidal cells: slow prepotentials dominate the primary range firing, Exp. Brain Res., 53 11984) 431 443. 8 Leung, L.S., Hippocampal electrical activity following local tetanization. 1. Afterdischarges, Brain Res., 419 (1987) 173 187. 9 Leung, L.S. and Shen, B., Hippocampal CA1 evoked response and radial 8-arm maze performance after hippocampal kindling, Brain Res., 555 (1991) 353-357. 10 Leung, L.S. and Yim, C.Y., Intrinsic membrane potential oscilla-
13
14
15
16
17
18
19
20
21
22
tions in hippocampal neurons in vitro, Brain Res., 553 (1991) 261274. Masukawa, L.M., Bernard, L.S. and Prince, D.A, Variations in electrophysiological properties of hippocampal neurons in different subfields, Brain Res., 242 (1982) 34t-344. McNamara, J.O., Bonhaus, D.W., Shin, C., Crain, B.J., Gellman, R.L. and Giacchino, J.L., The kindling model of epilepsy: a critical review, CRC Crit. Rev. Neurobiol., 1 (1985) 341-391. Mody, I., Stanton P.K. and Heinemann U., Activation of Nmethyl-D-aspartate receptors parallels changes in cellular and synaptic properties of dentate gyrus granule cells after kindling, J. Neurophysiol., 59 (1987) 1033 -1054. Muller, D. and Lynch, G., Evidence that changes in presynaptic calcium currents are not responsible for long-term potentiation in hippocampus, Brain Res., 479 (1989) 290-299. Oliver, M.W, and Miller, J.J., Inhibitory process of hippocampal CAI pyramidal neurons following kindling-induced epilepsy in the rat, Can. J. Physiol. Pharmacol., 63 11985) 872 878. Rocca, W.A., Sharbrough, F.W., Hauser, W.A., Annegers, J.F. and Schoenberg, B.S., Risk factors for complex partial seizures: a population-based case-control study, Ann. Neurol., 21 (1987) 22-3l. Sloviter, R.S, Permanenty altered hippocampal structure, excitability, and inhibition after experimental status epilepticus in the rat: the 'dormant basket cell' hypothesis and its possible relevance to temporal lobe epilepsy, Hippocampus, 1 11991) 41 66. Wadman, W.J., Heinmann, U., Konnerth, A. and Neuhaus, S., Hippocampal slices of kindled rats reveal calcium involvement in epileptogenesis, Exp. Brain Res., 57 11985)404-41)7. Wong, R.K.S., Prince, D.A. and Basbaum, H.I., lntradendritic recordings from hippocampal neurons, Proc. Natl. Acad. Sci. LISA, 76 (1979) 986- 990. Yamada, N. and Bilkey, D.K., Kindling-induced persistent alterations in the membrane and synaptic properties of CAI pyramidal cells, Brain Res., 561 (199t) 324~331. Zhao, D. and Leung, L.S., Effects of hippocampal kindling on paired-pulse response in CA1 in vitro, Brain Res., 564 (1991) 220 229. Zhao, D. and Leung, L.S, Hippocampal kindling induced pairedpulse depression in the dentate gyrus and paired-pulse facilitation in CA3, Brain Res., 582 11992) 163-167.
191
NSL 09520
Partial hippocampal kindling increases paired-pulse facilitation and burst frequency in hippocampal CA1 neurons Dichen Zhao and L. Stan Leung Departments of Clinical Neurological Sciences and Physiology, University of Western Ontario, London, Ontario, Canada (Received 4 December 1992; Revised version received 22 February 1993; Accepted 22 February 1993)
Key words: Kindling; Intracellular recording; Afterhyperpolarization; Membrane property; Presynaptic facilitation For up to 3 weeks after 15 evoked afterdischarges (partial kindling) in the hippocampus in vivo, paired-pulse facilitation of the CAI apical dendritic excitatory postsynaptic potentials (EPSPs), recorded from single neurons in vitro, was significantly larger in neurons of kindled than control rats. Partial kindling did not significantly affect the resting membrane potential, the threshold or size of the action potential (AP), the fast afterhyperpolarization, input resistance, time constant or the EPSP threshold. The number of APs induced within the initial 20 ms of a long-duration 0.5-nA depolarizing current was significantly higher in the kindled than control neurons. The increase in paired-pulse facilitation and intrinsic spiking in hippocampal CA1 after partial kindling may contribute to an increase in seizure susceptibility.
Seizures are known to cause long-lasting changes. Febrile seizure may induce complex partial seizures in adults [16]. Experimentally induced afterdischarges in the brain, if delivered repetitively (the kindling paradigrn) also induced persistent seizure susceptibility [3, 12]. The underlying cause of the kindling-induced seizure susceptibility is not known. In the hippocampus, it may be caused by an increase in the excitatory postsynaptic potentials (EPSPs) [1, 4, 9, 12, 13, 21], increased Ca 2÷ influx [18], loss of inhibition [4-6, 17, 21, 22] or increased intrinsic neuronal bursting [6, 12, 20]. The present study is part of an on-going effort to understand the physiological changes induced by seizures, using kindling as the experimental model. The rationale was to test the hypothesis that kindling induced an increase in the paired-pulse facilitation of the EPSPs in single cells of CA1 in vitro, as suggested by previous studies on the population EPSPs in CA1 [4, 6, 21]. The kindling was partial, i.e., delivered to a stage before generalized convulsions, to reveal the mechanisms involved in the early stage of epileptogenesis. The surgical, kindling and in vitro slice preparation procedures have been reported elsewhere [8, 9]. Briefly, kindling was delivered through chronic-indwelling electrodes implanted in stratum moleculare of hippocampal Correspondence. L.S. Leung, Department of Physiology, Health Sciences, University of Western Ontario, Medical Sciences Building, London, Ontario N6A 5A5, Canada. Fax: (1) (519) 6613827.
CA1 of male Long Evans rats. Experimental rats were given the kindling stimulus (1 s, 100 Hz, 0.1-ms square pulses) while control rats were given low-frequency stimulations (LFSs), each consisting of 100 pulses at 0.167 Hz. 15 ADs/LFSs were delivered hourly, 5/day over 3 days. On days 1-3 (day-1 group) or 21-23 (day-23 group) after the last day of kindling or control treatment, 500¢tm-thick slices were obtained from the middle part of the stimulated hippocampus of each rat [10, 21]. In some experiments, hippocampal slices were obtained from Wistar rats which were not chronically implanted with electrodes (the unstimulated control group). The in vitro slice was perfused with artificial cerebrospinal fluid of the after composition (mM): NaCI, 124; KC1, 5; NaH2PO4.H20, 1.25; MgSO4"7H20, 2; CaC12" 6H20, 2; NaHCO3, 26; and glucose, 10. A concentric bipolar stimulating electrode was placed at the Schaffer collaterals (stratum radiatum in CA1, on the CA3 side). CA1 neurons were recorded at the cell body layer [resting membrane potential of < -60 mV and overshooting action potentials (APs)] using micropipettes filled with 3 M potassium acetate. The potentials were amplified by an Axon-IA probe, digitized (at 10 KHz) and stored on line by a custom program [10]. A holding hyperpolarization current (< 0.19 nA) was sometimes necessary to suppress spontaneous firing. Input resistance (Rin), time constant, spike height were measured as described elsewhere [10]. The fast afterhyperpolarization (AHP) was measured at 1-5 ms after the spike peak. Paired-pulse stimulations
192
A
were given to the Schaffer collaterals at 0.1 Hz and interpulse intervals (IPIs) of 10- 200 ms. At least two stimulus intensities were used, one just below and one just above the spike threshold of the first pulse• The slope of the EPSP was measured over a 1.5-ms interval near the onset of the EPSP, which was typically at 1.5-2 ms from the shock artifact. The points used for the EPSP measure were clearly before the onset of the spike, for the first (El) or second pulse (E2). ANOVA and non-parametric (Wilcoxon) tests were used for testing statistical significance, using the number of neurons as the independent variable. All kindled rats remained at seizure stage I after 15 ADs, which was expected from a previous study [8]. 72 neurons were impaled in slices derived from 22 rats (Table I). There was no significant difference between neurons from kindled and control rats in the resting membrane potential, input resistance, time constant, the height, voltage and current threshold of the AP and fast AHP (Table I). The electrophysiological characteristics were typical of CAI pyramidal cells [10]. When injected with a long-duration depolarizing current, the typical firing pattern of CA1 neurons was a series of spikes that gradually decreased their interspike intervals (Fig. 1A). In some cells, however, a clustering of the early spikes (bursting) superimposed on a slow depolarizing hump was observed (Fig. IB). Some bursting cells were found in kindled and control groups and the occurrence of bursting neurons was only statistically different (P < 0.05, Z 2) in kindled than in control slices for day 1 but not for day 23. Except for a lower spike threshold current in bursting than non-bursting neurons, the membrane properties listed in Table I were not significantly different between the eight bursting and seven non-bursting neurons in the day-1 kindled group. However, we found it difficult to categorize bursting in some
-~
75ms~1
B
-j C
2.5~--
daylc - daylk
• .z~.
day23c
2.-
"~
1.5-
,<
~ ~
.5-
z'0
g0
~,.~.~ -.~,=.~_~
d0
8b ld0 1~0 110 td0
l a t e n c y a f t e r c u r r e n t injection (ms)
Fig. 1. Kindling induced changes in neuronal firing pattern. A,B: Examples of firing patterns induced by 2-3 times the threshold intensity of a long-duration (170-210 ms) depolarization current. A: A non-bursting neuron from a kindled day-23 slice; B: A bursting neurons from a kindled day-I rat. C: AP number/20-ms interval at various latencies after a 0.5-nA depolarization current.
CA1 neurons since the clustering of spikes and the slow hump may not be as clear as in Fig. 1. Thus, we quantified the spike frequency over short periods of time (20 ms) after the onset of a 0.5-nA depolarizing current. As expected, the spike frequency declined with time (accommodated) after the current onset [7]. The firing frequency for the initial 20 ms was significantly higher in the kindled than control neurons (Fig. 1C; Wilcoxon, P < 0.05,
TABLE I M E M B R A N E P R O P E R T I E S O F CAI P Y R A M I D A L CELLS F R O M K I N D L E D A N D C O N T R O L RATS Day 1
Day 23
Kindled
Control
Kindled
Control
Non-stimulated
N u m b e r of rats N umber of cells Resting potential (mV) Input resistance (MI2) Time constant (ms) Spike height(mV) Spike threshold (nA) Spike threshold (mV) A H P (mV)
8 15 71.7 47.1 11.1 72.0 0.20 12.67 2.5
6 18 70.2 48.8 12.4 70.6 0.29 14.87 2.57
4 16 72.9 43.0 10.5 77.5 0.18 11.1 2.15
4 12 74.5 45.2 11.9 71.4 0.26 15.32 2.4
Percent of bursting cells (%)
53.5
7 11 69.8 44.5 12.7 67.6 0.24 15.1 1.35 27.3
+_ 1.9 + 2.4 _+0.7 -+2.2 _+ 0.06 _+ 2.31 -+ 0.67
22.2
+_ 1.37 _+ 3.11 +0.7 + 1.43 _+ 0.05 -+ 1.42 -+ 0.61
12.5
+ 1.1 + 2.8 -+0.9 _+ 1.6 _+ 0.05 + 0.71 _+ 0.44
16.7
+ 1.6 _+ 2.7 -+1.2 _+ 2.6 + 0.07 + 1.97 +_ 0.45
_+ 2.5 _+ 6.9 _+ 1.4 +_ 3.0 + 0.06 +_ 1.9 _+ 0.12
193 for day 1 or 23 kindled vs. their respective controls) but not significantly different between different (day-1 or day-23) kindled groups or among different control groups. The stimulus threshold for evoking an AP in CA 1 neurons (< 30 I2A and < 0.15 ms duration) was not significantly different between control and kindled neurons. The synaptic responses evoked by stimuli at just above and just below the spike threshold were practically the same, whether a first spike (FS) or no FS was evoked (Fig. 2). The ratio of the slope of the EPSP evoked by E2 to that evoked by El, E2/E1, was significantly larger in kindled than control neurons, on either day 1 or 23 (Fig. 2). In a two-factor (experimental group x IPI) repeated measure ANOVA, there was a significant difference in E2/EI between kindled and control groups (P < 0.01 for all F evaluated between kindled and control groups for the four conditions: no FS-day 1, yes FS-day 1, no FSday 23 and yes FS-day 23t. There was no significant interaction between IPI and experimental group. E2/E1 was not different between yes FS and no FS groups and E1 was not significantly different among kindled and control groups. Previous studies reported that no changes were observed in the resting membrane potential, input resistance, spike amplitude and time constant of CA1 neurons after full kindling (generalized convulsions) of the commissure or perforant path [15, 20]. We confirmed the latter result using partial kindling. In addition, we observed a significant increase in the intrinsic excitability of CA1 neurons as tested by a depolarizing current. This was somewhat similar to the observation of a large number of bursting cells in 'kindled' slices by Yamada and Bilkey [20], though we, and others, also observed a large number of bursting CA 1 cells in control (low-frequency stimulated or unstimulated) slices [10, 11, 19]. We confirmed that kindling induced a significant increase in paired-pulse facilitation of the intracellularly recorded EPSPs. This facilitation was equally strong on days 1 and 23 after kindling (Fig. 2) and did not depend on whether the first pulse evoked a spike or not, i.e., E2 was minimally dependent on the A H P at - I 0 200 ms after the spike (which could reach several mV in amplitude). Paired-pulse EPSP facilitation may be caused by a presynaptic facilitation [2, 14] or a loss of postsynaptic (feedforward or feedback) inhibition. Our preliminary analysis of the neuronal hyperpolarization after low intensity afferent stimulation revealed no statistical difference between kindled and control groups but this presumed measure of the inhibition is tempered by other events (e.g., late EPSPs). The intracellular measure of the EPSP facilitation was found to be robust. The variance of intracellular E2/E1
FS
A 1.8-
1.4-
.C
-50
6
5'o
16o
1go
260
2~o
•.o- k-noFS v. k-FS -6-c-noFS --~ c-FS
B 1.8-
1.4-
.G
-s0
6
5'0
t60
~o
260
2~0
IPI (ms) Fig. 2. Inset on right top: Overlaid pair-pulse responses at a stimulus intensity that slightly exceeded the threshold of the FS and slightly below the FS threshold (no FS). IPI = 40 ms, from a control slice. Note the EPSP slopes and latency of the spike evoked by the second pulse were not affected by the presence or absence o f F S . E2/EI as a function of the IPI was plotted for day-I (A) and day-23 groups (B). In each graph, there are four subgroups: kindled no FS (k-noFS), kindled FS (k-FS), control no FS (c-noFS) and control FS (c-FS). For clarity, error bars for the four graphs are not included. N u m b e r of neurons (kindled no FS, kindled FS, control no FS and control FS): 12,10, 13,14 (A); 15,16, 17,17 (B).
was typically smaller than that of the extracellular (population) EPSPs [21], though the means of the extracelluar and intracellular EPSP facilitation was about the same. The use of low stimulus intensities in this study (~1.2 times the EPSP threshold) was made possible by the large signal to noise ratio of intracellular recordings. The kindling-induced changes reported here will tend to make CA1 more excitable and may contribute to seizure susceptibility. EPSP facilitation was shown to sum up during repeated pulses and may be an important factor in the spread and synchronization of large areas of
194
the b r a i n d u r i n g seizures. T h u s , s h o r t - t e r m plasticity, in a d d i t i o n to l o n g - t e r m p o t e n t i a t i o n [1, 12], s h o u l d be furt h e r s t u d i e d as a m e c h a n i s m a n d h u m a n seizures.
underlying experimental
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12 We t h a n k B. S h e n for t e c h n i c a l assistance. T h e research was supported by NS-25383, NSERC H o s p i t a l for Sick C h i l d r e n F o u n d a t i o n 91 --074.
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