270
Neuroscience Letters, 137 (1992) 270-274 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08480
Investigation offl-adrenergic modulation of synaptic transmission and postsynaptic induction of associative LTP in layer V neurones in slices of rat sensorimotor cortex Alex V. Nowicky, Geri Christofi a n d Lynn J. B i n d m a n Department of Physiology, University College London, London (UK) (Received 18 June 1991; Revised version received 16 December 1991 ; Accepted 16 December 1991)
Key words." Noradrenaline; Isoprenaline; Excitatory postsynaptic potential; Long-term potentiation Long-term potentiation (LTP) of synaptic transmission is considered to be a neuronal model of learning. Recently, the probability of induction of associative LTP in layer V cells in sensorimotor neocortex was shown to be much higher in the awake cat than in the slice preparation. We hypothesised that the loss of extrinsic noradrenergic activity in the slice might account for this difference, particularly since a fl-adrenergic enhancement of field potentials has been seen in this preparation. We therefore bath-applied noradrenaline (NA) or the flradrenergic agonist, isoprenaline (ISO) to elucidate the cellular basis of the enhancement of field potentials, and to see if the drugs increased the probability of induction of associative LTP in slices. We found that NA and ISO produced a dose-dependent, reversible reduction of spike accommodation and an increase in excitability but had no effect on the depolarizing slope or peak amplitude of sub-threshold EPSPs, and that drug application did not increase the probability of induction of LTP. We conclude that: (1) the enhancement of field potentials and late components of EPSPs (7) can be explained by the known actions offl-adrenergic drugs on membrane currents in layer V cells, and (2) the lower probability of induction of associative LTP in slices cf. the awake cat cannot be due solely to the loss of noradrenergic activity.
Noradrenergic projections to the forebrain are thought to be important in modulating synaptic activity in cortical circuits, particularly with respect to developmental and synaptic plasticity [4, 12, 16, 17]. However it is clear that noradrenaline (NA) produces different effects in different regions of the cortex [13, 14, 22]. In both the dentate and the CA3 region of hippocampus, fl-adrenergic actions produce a persistent increase in excitatory field potentials and population spikes [9, 15]. In CA1, NA and isoprenaline (ISO) produce a persistent enhancement of field EPSP to population spike (E-S) coupling, i.e. in the population spike evoked by a given stimulus, but not in the preceding excitatory field potential [14]. Both ~- and fl-adrenergic receptor subtypes are involved in modulating neocortical spike and field potential activity [1, 7, 19, 21, 23, 24]. Intracellular studies in both hippocampal and neocortical slices have shown that postsynaptic fl-adrenergic receptors mediate the reduction of Ca 2+- and Na*-dependent potassium currents, and in layer V cells of the cat somatosensory cortex NA also produces an increase in the persistent Correspondence: A.V. Nowicky, Department of Physiology, University College London, Gower St., London WC1E 6BT, UK.
(slow) Na ÷ current [10, 20]. However, no detailed intracellular analysis of immediate or long-term actions of NA and ISO on recorded synaptic potentials has been carried out in the neocortex. We have now carried out this analysis on the synaptic response to white matter stimulation in layer V neurones of the rat sensorimotor cortical slice. Coronal slices of sensorimotor cortex, 400/~m thick, were prepared from 35 Sprague-Dawley rats (I 60-190 g) and maintained in a standard interface recording chamber according to previously published methods [5]. Standard intracellular recording and stimulating techniques were used. Current pulses (-0.2 to -0.5 nA, 100 ms, every 10 s) were applied to monitor input resistance. After stable recording conditions were established, a 1015 min control recording was obtained prior to drug application or attempted induction of LTP. Accommodation of spike firing was tested with 500 ms depolarizing pulses throughout the experiment. Excitability was measured periodically as the current needed to evoke an action potential (100 ms, 0.1 nA up to 2 nA). Subthreshold EPSPs were evoked by stimulating white matter of the corpus callosum with bipolar electrodes (0.1 ms, 5-30 V, every 10 s). In some experiments, shocks of increased duration (0.2 ms to 0.5 ms) were used to elicit supra-
271
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20 uM ISO
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Fig. 1. Digitized responses of blockade of accommodation observed following 20 ktM ISO application to bathing medium. Upper traces in each pair, voltage; lower traces, current. Control: 0.7 nA, 500 ms depolarizing pulse to the cell produced 7 spikes. After 10 min of 20/~M ISO the same current produced 20 spikes. Cell resting potential was - 7 5 mV before and - 7 3 mV in ISO. R~, 23 MO. The blockade of accommodation was most pronounced in cells which depolarized 2-5 mV with N A or ISO, although it was still evident in cells in which the resting potential did not change. Calibration bars 100 ms, 25 mV and 1.5 nA.
threshold responses. The protocol used for the associative induction of LTP was as in ref. 5. LTP was defined as a significant increase in EPSP slope and/or peak amplitude at 30 min post-pairing. Drugs were obtained from Sigma and were diluted to final concentrations in ACSF. Five to 20 ~tM NA, or 10 to 20 HM ISO was perfused for up to 30 min followed by re-perfusion with normal ACSF. Blockade of accommodation was used to ascertain drug action on the recorded cell; the criterion was > 50% increase in the number of spikes elicited by the depolarizing pulse. Statistical comparisons were performed on averages of EPSPs (n=16) at various times post-treatment, using a one-way ANOVA. The data of EPSP slopes (initial 1-2 ms) and peak amplitudes were normalized and expressed as percentages of control values, using the formula: post/pre x 100%. The results of the experiments are based on stable intraceUular recordings from 41 layer V neurons, from 41 slices. They had the following characteristics: (1) cell location 54%+5% (mean +1 S.D.) of the depth of the grey matter below the pial surface, (2) mean resting potential -75+4.5 mV, (3) mean input resistance 23+5.8 Mr2, (4) mean action potential amplitude (from threshold to peak) 78+5.4 mV, and (5) spike accommodation exhibited by 37 out of 41 cells (90%). The subthreshold EPSPs had a mean peak amplitude in the control period
of 8.6+_3.3 mV, a mean onset latency of 2.8+-1.10 msec, and a mean peak latency of 8.7+-2.71 ms. The EPSPs were usually followed by an IPSP, revealed by depolarizing current but often reversed at resting potential. Rarely, a late hyperpolarizing IPSP was seen. Blockade of accommodation occurred within 10 min of drug application, and was dose-dependent: in 5/~M NA, 1 out of 7 cells (14%); 10/.tM NA, 2 out of 8 (25%); 20/~M NA, 9 out of 12 (75%); 10/.tM ISO, 5 out of 7 (71%) and 20/.tM ISO, 5 out of 6 (83%). The NA induced blockade was reversible within 15 to 20 min of re-perfusion with normal ACSF, but reversal of the ISO effects required >45 min. Hyperpolarization of 2-4 mV was seen in 25% of the cells, no change in resting potential in 33%, and depolarization from 2 to 5 mV in 42%. No significant effects on cell input resistance were found after exposure to 20/.tM NA, or 10 or 20/.tM ISO (control: 18.6 MI2+l.6 S.E.M.; 20 rain post-drug: 20.0 MO+ 1.8, F2,22=0.65). No significant effects of the drugs on EPSP slope or peak amplitude were observed 30 min after onset of drug perfusion, even at doses producing marked block of accommodation (see for example Fig. 3A). The means (+ 1 S.E.M. of control values) for the various drug doses were: 5/tM NA, 95.6% +-3.7% (n=5); 10/.tM NA, 89.0% +3.4% (n=5); 20/.tM NA, 104.0% +-11.9% (n=10), and for 10/IM ISO, 92.3% +-3.8% (n=5), and for 20/1m ISO, 115.6% +-6.4% (n=6) (/;'4.26=1.08). Similarly, no significant changes were seen in peak amplitudes; F4.26=0.33. Following 45 min of reperfusion of ACSF there were no significant differences in slope (F3m=0.31) or peak amplitude (F3m=0.93). In some experiments there were drug-induced effects on the EPSP duration, or on the spike latency in suprathreshold responses. In 4 cells (1 in 20/.tM NA and 3 in 20 HM ISO) we observed an increase in EPSP duration, accompanied by a depolarization of 1 to 4 mV in 2 of the cells and no change in the other 2./%Adrenergic actions on E-S coupling were assessed on sub-threshold and suprathreshold responses in 6 neurones (3 in 20/IM NA and 3 in 10/.tM ISO). A decreased spike latency was observed in 1 cell but this was accompanied by depolarization of 5 inV. In 2 other neurones there was a reduced spike latency after exposure to drug, accompanied by hyperpolarization but no change in initial depolarizing slope. In 3 cells there was no drug effect on the spike latency or on the EPSP slope of suprathreshold responses, although accommodation was blocked in each case. We assessed the probability of postsynaptic induction of LTP in 15 cells bathed in normal ACSF: LTP was induced in 2 out of 15 cells (13%) (Fig. 2 upper waveforms). We then examined possible drug actions in two
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10 ms Fig. 2. Digitized waveforms from two experiments illustrating associative LTP in ACSF or 20/tM NA, superimposing the baseline responses (dashed line) and the potentiated responses at 30 min (solid line). (1) Averaged EPSP (n-8) before and 30 min after successful LTP induced by associative pairing in ACSF. Conditioning current pulses !.2 nA, 150 ms, eliciting 8 spikes/pulse, applied 5 ms after onset of test shock, 50 pairings. Note increase of 30% in peak EPSP amplitude but not in initial depolarizing slope, followed by reduction of early (17 ms peak latency) hyperpolarizing IPSP. Late IPSP (110 ms peak latency) also r e d u c e d but not seen on this timebase. Cell repolarized in LTP to control resting potential of -70 mV for comparison. Rm 50 Mr2. Calibration bars 10 ms and 5 mV. (2) Averaged EPSP from another cell in w h i c h LTP was produced in 20/IM NA. Conditioning current pulses, 1.5 nA, 300 ms, eliciting 20-22 spikes/pulse, applied 20 ms prior to test shock, 50 pairings. Note increase of 27% in peak amplitude. The 75% increase in depolarizing slope was seen from 6 ms after onset of EPSP. Membrane potential unchanged after induction of LTP, at -68 mV. &,, 15 Mr2. Calibration bars 10 ms and 10 mV.
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series of experiments. In the first, 20 ~tM NA (n=2) or 20 pM ISO (n=2) was perfused for 30 min and then 1 or more successive conditioning attempts with increasing current strength were carried out at 30 min intervals. In 1 out of the 4 neurones, an increase in depolarizing slope and peak amplitude was produced by the third pairing in 20/aM NA (Fig. 2, lower waveforms). The increase in the depolarizing slope of the EPSP was delayed by 6 ms after the onset of the potentiated EPSP (see ref. 6). In 10 cells, the probability of induction of associative LTP was compared before and during continuous drug perfusion (see Fig. 3). Associative LTP was not induced following the first pairing attempt (PI) in normal ACSF (at 30 min, mean peak amplitudes 94.2_+3.5% of control values, n=10). After 30 min perfusion of either 20/tM NA (n=5) or 20 ~tM ISO (n=5), a second pairing attempt (PII) was undertaken using the same pairing parameters (in 20 /tM NA, 97.7+6.7%, and in 20 pM ISO, 101.4-+3.9%). Similarly, no associative LTP was observed at 30 min post-PII. A comparison of the mean peak values at 30 min after PI and PII for the two groups revealed no significant differences ( F 3 , 1 6 - - 0 . 2 5 ) . In summary, despite a doubling in the mean number of
Fig. 3. Representative waveforms and time course of an experiment involving the a t t e m p t e d induction of associative LTP before and during 20/lM ISO perfusion. A: superimposed averaged EPSP (n=8) at (1) pre-pairing period PI and 25 min post-PI, (2) Pre- and 25 min post-20 /IM ISO perfusion, (3) pre-second pairing period PII, and 35 min postPII. Dashed lines indicate pre-treatment, solid lines indicate post-treatment. R~, 20 MO. Calibration bars: 10 ms and 5 mV. B: plot of experimental time course of averaged (n=8) peak amplitudes of EPSPs (filled circles) and initial depolarizing slopes (filled triangles). Vertical lines indicate PI and PII, and horizontal bar the duration of perfusion of 20 /tM ISO.
spikes elicited per pairing train in the presence of the drugs (with 20/lM NA, 220% of control firing rate, n--7; and with 20/.zM ISO, 205% of control, n=7) the likelihood of postsynaptic induction of associative LTP was not increased in the presence of either drug (1 out of 14, 7%) compared with the i n c i d e n c e in A C S F (2 out of 15, 13%).
The c o n s i s t e n t l a c k of effect of N A o r I S O o n the depolarizing slope and peak amplitude of EPSPs evoked by white matter stimulation in the cells of this study suggests there is no presynaptic fl-adrenergic action on transmitter release at these synapses on layer V cells in sensori-
273 m o t o r cortex; in c o n t r a s t to synapses in the d e n t a t e gyrus where fl-adrenergic activity increases p r e s y n a p t i c glutam a t e release, a n d e n h a n c e s field E P S P s [18]. O u r i n t r a c e l l u l a r s t u d y o f N A a n d I S O on s y n a p t i c p o t e n t i a l s indicate t h a t the reversible e n h a n c e m e n t o f n e o c o r t i c a l field p o t e n t i a l s [7, 19] c o u l d have resulted f r o m one o r m o r e o f the following: (i) the d e p o l a r i z a t i o n o f the Vm l e a d i n g to earlier spike firing, (ii) i n c r e a s e d n e u r o n a l excitability, l e a d i n g to r e d u c e d spike latency a n d a n increase in firing in the p o p u l a t i o n o f d e p o l a r i z e d cells, a n d (iii) i n c r e a s e d firing rate due to r e d u c t i o n o f p o t a s s i u m currents, w h i c h c o u l d e n h a n c e di- a n d p o l y s y n a p t i c t r a n s m i s s i o n a l o n g the afferent p a t h w a y within the slice. Thus, the o b s e r v e d s h o r t - t e r m d r u g effects on the layer V s y n a p t i c responses r e s e m b l e d those in the CA1 region, b u t we d i d n o t see a l o n g - l a s t i n g fl-adrenergic e n h a n c e m e n t o f E-S c o u p l i n g [14] n o r d i d we find an increased p r o b a b i l i t y o f i n d u c t i o n o f LTP. We used a p o s t s y n a p t i c p r o t o c o l for i n d u c t i o n o f L T P i n s t e a d o f tetanic stimulus trains for t w o reasons. Firstly, this was the e x p e r i m e n t a l p a r a d i g m p r o d u c i n g i n d u c t i o n o f L T P in 50 to 60% o f c o n d i t i o n e d E P S P s in a w a k e a n i m a l s [3]. Secondly, p o s t s y n a p t i c injection o f E G T A b l o c k s posts y n a p t i c i n d u c t i o n o f L T P [2] suggesting t h a t L T P r e c o r d e d in the injected cell is a localized effect o f p o s t s y n a p t i c d e p o l a r i z a t i o n on the s y n a p t i c terminals. Posts y n a p t i c c o n d i t i o n i n g t h e r e f o r e allows a m o r e selective analysis o f n e o c o r t i c a l p h e n o m e n a t h a n tetanic stimulation o f afferents, b u t these results s h o u l d n o t be e x t r a p o lated to o t h e r n e o c o r t i c a l synapses. We c o n c l u d e t h a t the lower incidence o f L T P induct i o n o b s e r v e d in vitro when c o m p a r e d to t h a t in vivo is n o t due simply to a l a c k o f n o r a d r e n e r g i c activity in the slice [2, 3, 5]. It m a y be t h a t o t h e r n e u r o m o d u l a t o r s assist in the p r o d u c t i o n o f n e o c o r t i c a l associative L T P [8]. We t h a n k T h e W e l l c o m e T r u s t for financial s u p p o r t o f A.V.N. a n d the research. G . C . is a n M . R . C . scholar. 1 Armstrong-James, M. and Fox, K., Effects of iontophoresed noradrenaline on the spontaneous activity of neurones in rat primary somatosensory cortex, J. Physiol., 335 (1983) 427-447. 2 Baranyi, A. and Szente, M.B., Long-lasting potentiation ofsynaptic transmission requires postsynaptic modifications in the neocortex, Brain Res., 423 (1987) 378-384. 3 Baranyi, A., Szente, M.B. and Woody, C.D., Properties of associative long-lasting potentiation induced by cellular conditioning in the motor cortex of conscious cats, Neurosci., 42 (1991) 321-334. 4 Bear, M.F. and Singer, W., Modulation of visual cortical plasticity by aeetylcholine and noradrenaline, Nature, 320 (1986) 172-176. 5 Bindman, L.J., Murphy, K.P.S.J. and Pockett, S., Postsynaptic control of induction of long-term changes in efficacy of transmission at neocortical synapses in slices of rat brain, J. Neurophysiol., 60 (1988) 1053-1065. 6 Bindman, L.J. and Murphy, K.P.S.J,, Delayed onset of potentiation in neocortical EPSPs during long-term potentiation (LTP) - - a
postsynaptic mechanism or heterogeneous synaptic inputs?, Adv. Exp. Med. Biol., 268 (1990) 307-312. 7 Bindman, L.J., Dedicoat, M., Hamiduddin, R.B., Harnett, P.A. and Pegna, A., The effect of noradrenaline on synaptic transmission in slices of rat neocortex in vitro, J. Physiol., 423 (1990) 97P. 8 Brocher, S., Artola, A. and Singer, W., Norepinephrine and acetylcholine act synergisitically in facilitating LTP in the rat visual cortex, Eur. J. Neurosci., Suppl. 2 (1989) 18-19. 9 Dahl, D. and Sarvey, J.M., Norepinephrine induces pathway-specific long-lasting potentiation and depression in the hippocampal dentate gyms, Proc. Natl. Acad. Sci. USA, 86 (1989) 4776~780. 10 Foehring, R.C., Schwindt, P.C. and Crill, W.E., Norepinephrine selectively reduces slow Ca2÷ and Na ÷ mediated K + currents in cat neocortical neurons, J. Neurophysiol., 61 (1989) 245-256. 11 Gray, R. and Johnston, D., Noradrenaline and fl-adrenoeeptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons, Nature, 327 (1987) 620~522. 12 Harley, C.W., A role for norepinephrine in arousal, emotion, and learning?: Limbic modulation by norepinephrine and the Kety hypothesis, Prog. Neuropsychopharmacol. Biol. Psychiatry, 11 (1988) 419-458. 13 Harley, C.W., Milway, J.S. and Lacaille, J.C., Locus coeruleus potentiation of dentate gyrus responses: evidence for two systems, Brain Res. Bull., 22 (1989) 643~550. 14 Heginbotham and Dunwiddie, T.V., Long-term increases in the evoked population spike in the CA 1 region of the rat hippocampus induced by fl-adrenergic receptor activation, J. Neurosci., 11 (1991) 2519-2527. 15 Hopkins, W.F. and Johnston, D., Noradrenergic enhancement of long-term potentiation at mossy fiber synapse in the hippocampus, J. Neurophysiol., 59 (1988) 667~i87. 16 Lehmenkuhler, C., Walden, J. and Speckmann, E.J., Decrease of N-methyl-D-aspartate responses by noradrenaline in the rat motorcortex in vivo, Neurosci. Lett., 121 (1991) 5-8. 17 Levin, B.E., Craik, B.L. and Hand, P.J., The role of norepinephrine in adult rat somatosensory (SmI) cortical metabolism and plasticity, Brain Res., 443 (1988) 261-271. 18 Lynch, M.A. and Bliss, T.V.P., Noradrenaline modulates the release of [J4C]-glutamate from dentate but not from CA1/CA3 slices of rat hippocampus, Neuropharmacology, 25 (1986) 493-498. 19 Nowicky, A.V., Berry, R. and Teyler, T.J., Noradrenaline and isoprenaline produce reversible enhancement of synaptic responses in rat visual cortex, in vitro, J. Physiol., 424 (1990) 62P. 20 Madison, D.V. and Nicoll, R.A., Actions of noradrenaline recorded intracellularly in rat hippocampal pyramidal neurons, in vitro, J. Physiol., 372 (1986) 221-244. 21 Mouradian, R.D., Sessler, F.M. and Waterhouse, B.D., Noradrenergic potentiation of excitatory transmitter action in cerebrocortical slices: evidence for mediation by an ctl-receptor-linked second messenger pathway, Brain Res., 546 (1991) 83-95. 22 Sarvey, J.M., Burgard, E.C. and Decker, G., Long-term potentiation: studies in the hippocampal slice, J. Neurosci. Methods, 28 (1989) 109-124. 23 Waterhouse, B.D., Moises, H.C. and Woodward, D., Noradrenergic modulation of somatosensory cortical neuronal response to iontophoreticallyapplied putative neurotransmitters, Exp. Neurol,, 69 (1980) 30459. 24 Waterhouse, B.D., Moises, H.C., Yeh, H.H. and Woodward, D., Norepinephrine enhancement of inhibitory synaptic mechanisms in cerebellum and cerebral cortex: mediation by fl-adrenergic receptors, J. Pharm. Exp. Ther., 221 (1982) 495-505.
Neuroscience Letters, 137 (1992) 270-274 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08480
Investigation offl-adrenergic modulation of synaptic transmission and postsynaptic induction of associative LTP in layer V neurones in slices of rat sensorimotor cortex Alex V. Nowicky, Geri Christofi a n d Lynn J. B i n d m a n Department of Physiology, University College London, London (UK) (Received 18 June 1991; Revised version received 16 December 1991 ; Accepted 16 December 1991)
Key words." Noradrenaline; Isoprenaline; Excitatory postsynaptic potential; Long-term potentiation Long-term potentiation (LTP) of synaptic transmission is considered to be a neuronal model of learning. Recently, the probability of induction of associative LTP in layer V cells in sensorimotor neocortex was shown to be much higher in the awake cat than in the slice preparation. We hypothesised that the loss of extrinsic noradrenergic activity in the slice might account for this difference, particularly since a fl-adrenergic enhancement of field potentials has been seen in this preparation. We therefore bath-applied noradrenaline (NA) or the flradrenergic agonist, isoprenaline (ISO) to elucidate the cellular basis of the enhancement of field potentials, and to see if the drugs increased the probability of induction of associative LTP in slices. We found that NA and ISO produced a dose-dependent, reversible reduction of spike accommodation and an increase in excitability but had no effect on the depolarizing slope or peak amplitude of sub-threshold EPSPs, and that drug application did not increase the probability of induction of LTP. We conclude that: (1) the enhancement of field potentials and late components of EPSPs (7) can be explained by the known actions offl-adrenergic drugs on membrane currents in layer V cells, and (2) the lower probability of induction of associative LTP in slices cf. the awake cat cannot be due solely to the loss of noradrenergic activity.
Noradrenergic projections to the forebrain are thought to be important in modulating synaptic activity in cortical circuits, particularly with respect to developmental and synaptic plasticity [4, 12, 16, 17]. However it is clear that noradrenaline (NA) produces different effects in different regions of the cortex [13, 14, 22]. In both the dentate and the CA3 region of hippocampus, fl-adrenergic actions produce a persistent increase in excitatory field potentials and population spikes [9, 15]. In CA1, NA and isoprenaline (ISO) produce a persistent enhancement of field EPSP to population spike (E-S) coupling, i.e. in the population spike evoked by a given stimulus, but not in the preceding excitatory field potential [14]. Both ~- and fl-adrenergic receptor subtypes are involved in modulating neocortical spike and field potential activity [1, 7, 19, 21, 23, 24]. Intracellular studies in both hippocampal and neocortical slices have shown that postsynaptic fl-adrenergic receptors mediate the reduction of Ca 2+- and Na*-dependent potassium currents, and in layer V cells of the cat somatosensory cortex NA also produces an increase in the persistent Correspondence: A.V. Nowicky, Department of Physiology, University College London, Gower St., London WC1E 6BT, UK.
(slow) Na ÷ current [10, 20]. However, no detailed intracellular analysis of immediate or long-term actions of NA and ISO on recorded synaptic potentials has been carried out in the neocortex. We have now carried out this analysis on the synaptic response to white matter stimulation in layer V neurones of the rat sensorimotor cortical slice. Coronal slices of sensorimotor cortex, 400/~m thick, were prepared from 35 Sprague-Dawley rats (I 60-190 g) and maintained in a standard interface recording chamber according to previously published methods [5]. Standard intracellular recording and stimulating techniques were used. Current pulses (-0.2 to -0.5 nA, 100 ms, every 10 s) were applied to monitor input resistance. After stable recording conditions were established, a 1015 min control recording was obtained prior to drug application or attempted induction of LTP. Accommodation of spike firing was tested with 500 ms depolarizing pulses throughout the experiment. Excitability was measured periodically as the current needed to evoke an action potential (100 ms, 0.1 nA up to 2 nA). Subthreshold EPSPs were evoked by stimulating white matter of the corpus callosum with bipolar electrodes (0.1 ms, 5-30 V, every 10 s). In some experiments, shocks of increased duration (0.2 ms to 0.5 ms) were used to elicit supra-
271
__l
!
20 uM ISO
] - -
I
/ E5 mV 1.5 nA 100 m s _ _ ]
Fig. 1. Digitized responses of blockade of accommodation observed following 20 ktM ISO application to bathing medium. Upper traces in each pair, voltage; lower traces, current. Control: 0.7 nA, 500 ms depolarizing pulse to the cell produced 7 spikes. After 10 min of 20/~M ISO the same current produced 20 spikes. Cell resting potential was - 7 5 mV before and - 7 3 mV in ISO. R~, 23 MO. The blockade of accommodation was most pronounced in cells which depolarized 2-5 mV with N A or ISO, although it was still evident in cells in which the resting potential did not change. Calibration bars 100 ms, 25 mV and 1.5 nA.
threshold responses. The protocol used for the associative induction of LTP was as in ref. 5. LTP was defined as a significant increase in EPSP slope and/or peak amplitude at 30 min post-pairing. Drugs were obtained from Sigma and were diluted to final concentrations in ACSF. Five to 20 ~tM NA, or 10 to 20 HM ISO was perfused for up to 30 min followed by re-perfusion with normal ACSF. Blockade of accommodation was used to ascertain drug action on the recorded cell; the criterion was > 50% increase in the number of spikes elicited by the depolarizing pulse. Statistical comparisons were performed on averages of EPSPs (n=16) at various times post-treatment, using a one-way ANOVA. The data of EPSP slopes (initial 1-2 ms) and peak amplitudes were normalized and expressed as percentages of control values, using the formula: post/pre x 100%. The results of the experiments are based on stable intraceUular recordings from 41 layer V neurons, from 41 slices. They had the following characteristics: (1) cell location 54%+5% (mean +1 S.D.) of the depth of the grey matter below the pial surface, (2) mean resting potential -75+4.5 mV, (3) mean input resistance 23+5.8 Mr2, (4) mean action potential amplitude (from threshold to peak) 78+5.4 mV, and (5) spike accommodation exhibited by 37 out of 41 cells (90%). The subthreshold EPSPs had a mean peak amplitude in the control period
of 8.6+_3.3 mV, a mean onset latency of 2.8+-1.10 msec, and a mean peak latency of 8.7+-2.71 ms. The EPSPs were usually followed by an IPSP, revealed by depolarizing current but often reversed at resting potential. Rarely, a late hyperpolarizing IPSP was seen. Blockade of accommodation occurred within 10 min of drug application, and was dose-dependent: in 5/~M NA, 1 out of 7 cells (14%); 10/.tM NA, 2 out of 8 (25%); 20/~M NA, 9 out of 12 (75%); 10/.tM ISO, 5 out of 7 (71%) and 20/.tM ISO, 5 out of 6 (83%). The NA induced blockade was reversible within 15 to 20 min of re-perfusion with normal ACSF, but reversal of the ISO effects required >45 min. Hyperpolarization of 2-4 mV was seen in 25% of the cells, no change in resting potential in 33%, and depolarization from 2 to 5 mV in 42%. No significant effects on cell input resistance were found after exposure to 20/.tM NA, or 10 or 20/.tM ISO (control: 18.6 MI2+l.6 S.E.M.; 20 rain post-drug: 20.0 MO+ 1.8, F2,22=0.65). No significant effects of the drugs on EPSP slope or peak amplitude were observed 30 min after onset of drug perfusion, even at doses producing marked block of accommodation (see for example Fig. 3A). The means (+ 1 S.E.M. of control values) for the various drug doses were: 5/tM NA, 95.6% +-3.7% (n=5); 10/.tM NA, 89.0% +3.4% (n=5); 20/.tM NA, 104.0% +-11.9% (n=10), and for 10/IM ISO, 92.3% +-3.8% (n=5), and for 20/1m ISO, 115.6% +-6.4% (n=6) (/;'4.26=1.08). Similarly, no significant changes were seen in peak amplitudes; F4.26=0.33. Following 45 min of reperfusion of ACSF there were no significant differences in slope (F3m=0.31) or peak amplitude (F3m=0.93). In some experiments there were drug-induced effects on the EPSP duration, or on the spike latency in suprathreshold responses. In 4 cells (1 in 20/.tM NA and 3 in 20 HM ISO) we observed an increase in EPSP duration, accompanied by a depolarization of 1 to 4 mV in 2 of the cells and no change in the other 2./%Adrenergic actions on E-S coupling were assessed on sub-threshold and suprathreshold responses in 6 neurones (3 in 20/IM NA and 3 in 10/.tM ISO). A decreased spike latency was observed in 1 cell but this was accompanied by depolarization of 5 inV. In 2 other neurones there was a reduced spike latency after exposure to drug, accompanied by hyperpolarization but no change in initial depolarizing slope. In 3 cells there was no drug effect on the spike latency or on the EPSP slope of suprathreshold responses, although accommodation was blocked in each case. We assessed the probability of postsynaptic induction of LTP in 15 cells bathed in normal ACSF: LTP was induced in 2 out of 15 cells (13%) (Fig. 2 upper waveforms). We then examined possible drug actions in two
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10 ms Fig. 2. Digitized waveforms from two experiments illustrating associative LTP in ACSF or 20/tM NA, superimposing the baseline responses (dashed line) and the potentiated responses at 30 min (solid line). (1) Averaged EPSP (n-8) before and 30 min after successful LTP induced by associative pairing in ACSF. Conditioning current pulses !.2 nA, 150 ms, eliciting 8 spikes/pulse, applied 5 ms after onset of test shock, 50 pairings. Note increase of 30% in peak EPSP amplitude but not in initial depolarizing slope, followed by reduction of early (17 ms peak latency) hyperpolarizing IPSP. Late IPSP (110 ms peak latency) also r e d u c e d but not seen on this timebase. Cell repolarized in LTP to control resting potential of -70 mV for comparison. Rm 50 Mr2. Calibration bars 10 ms and 5 mV. (2) Averaged EPSP from another cell in w h i c h LTP was produced in 20/IM NA. Conditioning current pulses, 1.5 nA, 300 ms, eliciting 20-22 spikes/pulse, applied 20 ms prior to test shock, 50 pairings. Note increase of 27% in peak amplitude. The 75% increase in depolarizing slope was seen from 6 ms after onset of EPSP. Membrane potential unchanged after induction of LTP, at -68 mV. &,, 15 Mr2. Calibration bars 10 ms and 10 mV.
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series of experiments. In the first, 20 ~tM NA (n=2) or 20 pM ISO (n=2) was perfused for 30 min and then 1 or more successive conditioning attempts with increasing current strength were carried out at 30 min intervals. In 1 out of the 4 neurones, an increase in depolarizing slope and peak amplitude was produced by the third pairing in 20/aM NA (Fig. 2, lower waveforms). The increase in the depolarizing slope of the EPSP was delayed by 6 ms after the onset of the potentiated EPSP (see ref. 6). In 10 cells, the probability of induction of associative LTP was compared before and during continuous drug perfusion (see Fig. 3). Associative LTP was not induced following the first pairing attempt (PI) in normal ACSF (at 30 min, mean peak amplitudes 94.2_+3.5% of control values, n=10). After 30 min perfusion of either 20/tM NA (n=5) or 20 ~tM ISO (n=5), a second pairing attempt (PII) was undertaken using the same pairing parameters (in 20 /tM NA, 97.7+6.7%, and in 20 pM ISO, 101.4-+3.9%). Similarly, no associative LTP was observed at 30 min post-PII. A comparison of the mean peak values at 30 min after PI and PII for the two groups revealed no significant differences ( F 3 , 1 6 - - 0 . 2 5 ) . In summary, despite a doubling in the mean number of
Fig. 3. Representative waveforms and time course of an experiment involving the a t t e m p t e d induction of associative LTP before and during 20/lM ISO perfusion. A: superimposed averaged EPSP (n=8) at (1) pre-pairing period PI and 25 min post-PI, (2) Pre- and 25 min post-20 /IM ISO perfusion, (3) pre-second pairing period PII, and 35 min postPII. Dashed lines indicate pre-treatment, solid lines indicate post-treatment. R~, 20 MO. Calibration bars: 10 ms and 5 mV. B: plot of experimental time course of averaged (n=8) peak amplitudes of EPSPs (filled circles) and initial depolarizing slopes (filled triangles). Vertical lines indicate PI and PII, and horizontal bar the duration of perfusion of 20 /tM ISO.
spikes elicited per pairing train in the presence of the drugs (with 20/lM NA, 220% of control firing rate, n--7; and with 20/.zM ISO, 205% of control, n=7) the likelihood of postsynaptic induction of associative LTP was not increased in the presence of either drug (1 out of 14, 7%) compared with the i n c i d e n c e in A C S F (2 out of 15, 13%).
The c o n s i s t e n t l a c k of effect of N A o r I S O o n the depolarizing slope and peak amplitude of EPSPs evoked by white matter stimulation in the cells of this study suggests there is no presynaptic fl-adrenergic action on transmitter release at these synapses on layer V cells in sensori-
273 m o t o r cortex; in c o n t r a s t to synapses in the d e n t a t e gyrus where fl-adrenergic activity increases p r e s y n a p t i c glutam a t e release, a n d e n h a n c e s field E P S P s [18]. O u r i n t r a c e l l u l a r s t u d y o f N A a n d I S O on s y n a p t i c p o t e n t i a l s indicate t h a t the reversible e n h a n c e m e n t o f n e o c o r t i c a l field p o t e n t i a l s [7, 19] c o u l d have resulted f r o m one o r m o r e o f the following: (i) the d e p o l a r i z a t i o n o f the Vm l e a d i n g to earlier spike firing, (ii) i n c r e a s e d n e u r o n a l excitability, l e a d i n g to r e d u c e d spike latency a n d a n increase in firing in the p o p u l a t i o n o f d e p o l a r i z e d cells, a n d (iii) i n c r e a s e d firing rate due to r e d u c t i o n o f p o t a s s i u m currents, w h i c h c o u l d e n h a n c e di- a n d p o l y s y n a p t i c t r a n s m i s s i o n a l o n g the afferent p a t h w a y within the slice. Thus, the o b s e r v e d s h o r t - t e r m d r u g effects on the layer V s y n a p t i c responses r e s e m b l e d those in the CA1 region, b u t we d i d n o t see a l o n g - l a s t i n g fl-adrenergic e n h a n c e m e n t o f E-S c o u p l i n g [14] n o r d i d we find an increased p r o b a b i l i t y o f i n d u c t i o n o f LTP. We used a p o s t s y n a p t i c p r o t o c o l for i n d u c t i o n o f L T P i n s t e a d o f tetanic stimulus trains for t w o reasons. Firstly, this was the e x p e r i m e n t a l p a r a d i g m p r o d u c i n g i n d u c t i o n o f L T P in 50 to 60% o f c o n d i t i o n e d E P S P s in a w a k e a n i m a l s [3]. Secondly, p o s t s y n a p t i c injection o f E G T A b l o c k s posts y n a p t i c i n d u c t i o n o f L T P [2] suggesting t h a t L T P r e c o r d e d in the injected cell is a localized effect o f p o s t s y n a p t i c d e p o l a r i z a t i o n on the s y n a p t i c terminals. Posts y n a p t i c c o n d i t i o n i n g t h e r e f o r e allows a m o r e selective analysis o f n e o c o r t i c a l p h e n o m e n a t h a n tetanic stimulation o f afferents, b u t these results s h o u l d n o t be e x t r a p o lated to o t h e r n e o c o r t i c a l synapses. We c o n c l u d e t h a t the lower incidence o f L T P induct i o n o b s e r v e d in vitro when c o m p a r e d to t h a t in vivo is n o t due simply to a l a c k o f n o r a d r e n e r g i c activity in the slice [2, 3, 5]. It m a y be t h a t o t h e r n e u r o m o d u l a t o r s assist in the p r o d u c t i o n o f n e o c o r t i c a l associative L T P [8]. We t h a n k T h e W e l l c o m e T r u s t for financial s u p p o r t o f A.V.N. a n d the research. G . C . is a n M . R . C . scholar. 1 Armstrong-James, M. and Fox, K., Effects of iontophoresed noradrenaline on the spontaneous activity of neurones in rat primary somatosensory cortex, J. Physiol., 335 (1983) 427-447. 2 Baranyi, A. and Szente, M.B., Long-lasting potentiation ofsynaptic transmission requires postsynaptic modifications in the neocortex, Brain Res., 423 (1987) 378-384. 3 Baranyi, A., Szente, M.B. and Woody, C.D., Properties of associative long-lasting potentiation induced by cellular conditioning in the motor cortex of conscious cats, Neurosci., 42 (1991) 321-334. 4 Bear, M.F. and Singer, W., Modulation of visual cortical plasticity by aeetylcholine and noradrenaline, Nature, 320 (1986) 172-176. 5 Bindman, L.J., Murphy, K.P.S.J. and Pockett, S., Postsynaptic control of induction of long-term changes in efficacy of transmission at neocortical synapses in slices of rat brain, J. Neurophysiol., 60 (1988) 1053-1065. 6 Bindman, L.J. and Murphy, K.P.S.J,, Delayed onset of potentiation in neocortical EPSPs during long-term potentiation (LTP) - - a
postsynaptic mechanism or heterogeneous synaptic inputs?, Adv. Exp. Med. Biol., 268 (1990) 307-312. 7 Bindman, L.J., Dedicoat, M., Hamiduddin, R.B., Harnett, P.A. and Pegna, A., The effect of noradrenaline on synaptic transmission in slices of rat neocortex in vitro, J. Physiol., 423 (1990) 97P. 8 Brocher, S., Artola, A. and Singer, W., Norepinephrine and acetylcholine act synergisitically in facilitating LTP in the rat visual cortex, Eur. J. Neurosci., Suppl. 2 (1989) 18-19. 9 Dahl, D. and Sarvey, J.M., Norepinephrine induces pathway-specific long-lasting potentiation and depression in the hippocampal dentate gyms, Proc. Natl. Acad. Sci. USA, 86 (1989) 4776~780. 10 Foehring, R.C., Schwindt, P.C. and Crill, W.E., Norepinephrine selectively reduces slow Ca2÷ and Na ÷ mediated K + currents in cat neocortical neurons, J. Neurophysiol., 61 (1989) 245-256. 11 Gray, R. and Johnston, D., Noradrenaline and fl-adrenoeeptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons, Nature, 327 (1987) 620~522. 12 Harley, C.W., A role for norepinephrine in arousal, emotion, and learning?: Limbic modulation by norepinephrine and the Kety hypothesis, Prog. Neuropsychopharmacol. Biol. Psychiatry, 11 (1988) 419-458. 13 Harley, C.W., Milway, J.S. and Lacaille, J.C., Locus coeruleus potentiation of dentate gyrus responses: evidence for two systems, Brain Res. Bull., 22 (1989) 643~550. 14 Heginbotham and Dunwiddie, T.V., Long-term increases in the evoked population spike in the CA 1 region of the rat hippocampus induced by fl-adrenergic receptor activation, J. Neurosci., 11 (1991) 2519-2527. 15 Hopkins, W.F. and Johnston, D., Noradrenergic enhancement of long-term potentiation at mossy fiber synapse in the hippocampus, J. Neurophysiol., 59 (1988) 667~i87. 16 Lehmenkuhler, C., Walden, J. and Speckmann, E.J., Decrease of N-methyl-D-aspartate responses by noradrenaline in the rat motorcortex in vivo, Neurosci. Lett., 121 (1991) 5-8. 17 Levin, B.E., Craik, B.L. and Hand, P.J., The role of norepinephrine in adult rat somatosensory (SmI) cortical metabolism and plasticity, Brain Res., 443 (1988) 261-271. 18 Lynch, M.A. and Bliss, T.V.P., Noradrenaline modulates the release of [J4C]-glutamate from dentate but not from CA1/CA3 slices of rat hippocampus, Neuropharmacology, 25 (1986) 493-498. 19 Nowicky, A.V., Berry, R. and Teyler, T.J., Noradrenaline and isoprenaline produce reversible enhancement of synaptic responses in rat visual cortex, in vitro, J. Physiol., 424 (1990) 62P. 20 Madison, D.V. and Nicoll, R.A., Actions of noradrenaline recorded intracellularly in rat hippocampal pyramidal neurons, in vitro, J. Physiol., 372 (1986) 221-244. 21 Mouradian, R.D., Sessler, F.M. and Waterhouse, B.D., Noradrenergic potentiation of excitatory transmitter action in cerebrocortical slices: evidence for mediation by an ctl-receptor-linked second messenger pathway, Brain Res., 546 (1991) 83-95. 22 Sarvey, J.M., Burgard, E.C. and Decker, G., Long-term potentiation: studies in the hippocampal slice, J. Neurosci. Methods, 28 (1989) 109-124. 23 Waterhouse, B.D., Moises, H.C. and Woodward, D., Noradrenergic modulation of somatosensory cortical neuronal response to iontophoreticallyapplied putative neurotransmitters, Exp. Neurol,, 69 (1980) 30459. 24 Waterhouse, B.D., Moises, H.C., Yeh, H.H. and Woodward, D., Norepinephrine enhancement of inhibitory synaptic mechanisms in cerebellum and cerebral cortex: mediation by fl-adrenergic receptors, J. Pharm. Exp. Ther., 221 (1982) 495-505.
