(NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and a

Transcription

1 Journal of Physiology (1991), 433, pp With 8 figures Printed in Great Britain MECHANISMS UNDERLYING POTENTIATION OF SYNAPTIC TRANSMISSION IN RAT ANTERIOR CINGULATE CORTEX IN VITRO BY P. SAH* AND R. A. NICOLL From the Departments of Pharmacology and Physiology, University of California, San Francisco, USA (Received 3 January 199) SUMMARY 1. The properties of the excitatory synapse made by callosal inputs onto layer V and layer VI cells in the anterior cingulate cortex were studied in an in vitro slice preparation with intracellular recording. 2. In the presence of picrotoxin, the excitatory postsynaptic potential (EPSP) had two components, a fast component blocked by the non-n-methyl-d-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and a slow component blocked by the NMDA receptor antagonist DL-2-amino-5- phosphonovalerate (APV). 3. Delivery of a brief tetanus to the afferent fibres led to a long-term potentiation (LTP) of the initial slope of the monosynaptic EPSP. The LTP displayed the property of co-operativity and could be blocked by APV or by buffering intracellular calcium. 4. Pairing of low frequency presynaptic activity or weak tetanic stimulation with postsynaptic depolarization failed to potentiate the EPSP. This suggests that postsynaptic depolarization alone is unable to explain the co-operativity. 5. It is concluded that the transmitter mediating the excitatory input between callosal afferents and layer V and layer VI pyramidal neurones is glutamate. Tetanic stimulation of these afferents leads to LTP which shares many but not all the properties of LTP seen in the CAI region of the hippocampus. INTRODUCTION Long-term potentiation (LTP) is a long lasting increase in synaptic efficacy produced by brief repetitive activation of afferent fibres. The most detailed studies of LTP have been carried out at excitatory synapses made by Schaffer collateralcommissural fibres onto CAI pyramidal cells in the hippocampus, where it has been shown that LTP is produced by conjunction of presynaptic activity and postsynaptic depolarization (for review see Collingridge & Bliss, 1987; Nicoll, Kauer & Malenka, 1988; Wigstr6m & Gustaffson, 1988). This associative property of LTP as well as its * Present address: Department of Physiology and Pharmacology, The University of Queensland, Queensland 472, Australia. MS 81f63

2 616 P. SAH AND R. A. NICOLL long duration have made it an attractive model for memory storage and learning. LTP has also been demonstrated in several regions outside the hippocampus (Artola & Singer, 1987; Baranyi & Szente, 1987; Bindman, Meyer & Pockett, 1987; Sakamoto, Porter, & Asanuma, 1987; Perkins & Teyler, 1988; Racine, Milgram & Hafner, 1983; Komatsu, Fujii, Maeda, Sakaguchi, & Toyama, 1988; Rasmusson & Dykes, 1988; Stripling, Patneau & Gramlich, 1988; Sutor & Hablitz, 1989a, b). Although some of the properties of LTP in these regions resemble that seen in the hippocampus, the mechanisms involved are poorly understood. One of the problems in studying LTP in the cerebral cortex is the difficulty of stimulating a defined monosynaptic input. Most studies have been limited to stimulation of the underlying white matter which not only contain afferent axons from different sources (e.g. thalamic, commissural), but also leads to stimulation of excitatory interneurones. The dual problems of a mixed afferent input and production of polysynaptic activity makes interpretation of synaptic responses difficult. An in vitro preparation of the cingulate cortex has been described in which it is possible to preserve a monosynaptic input from the corpus callosum (Vogt & Gorman, 1982). In this paper we report some of the mechanisms involved in LTP at the monosynaptic connection between fibres in the corpus callosum and layer V and layer VI pyramidal cells in the anterior cingulate cortex. Some of these results have been presented in abstract form (Sah & Nicoll, 1989). METHODS Slices of rat anterior cingulate cortex were obtained as outlined by Vogt & Gorman (1982). Under halothane anaesthesia, male Sprague-Dawley rats were decapitated and the brain rapidly removed and placed into cold oxygenated Ringer solution of composition (in mm): NaCl, 115; KCl, 2-5; MgSO4, 113; CaCl2, 2-5; NaH2PO4, 1 2; NaHCO3, 25; dextrose, 1; ph 7-2 when bubbled with 95% 2/5 % CO2. The lateral cortices were then removed with two parasagittal cuts, and the brain stem removed with a rostrocaudal cut through approximately the level of the hippocampus and stratum. The anterior half of the brain was glued onto the stage of a vibratome and 4,um thick, bilateral, coronal sections were taken. Slices were allowed to recover for at least 1 h before recording was attempted. A single slice was then transferred to the recording chamber (Nicoll & Alger, 1981) where it was held completely submerged between two nylon nets. The chamber was continuously perfused with oxygenated Ringer solution of composition (mm): NaCl, 115; KCl, 5*; MgSO4, 4 ; CaCl2, 4 ; NaH.PO4, 1-2; NaHCO3, 25; dextrose, 1 and at a temperature of 28-3 'C. Picrotoxin (1 um) was present in all the experiments illustrated in this paper. Intracellular recordings were obtained from pyramidal cells in layer V and layer VI of the anterior cingulate cortex. Most of the cells in this study were of the regular spiking type (Connors, Gutnick & Prince, 1982) and many could be identified by antidromic invasion from the corpus callosum. Intracellular microelectrodes were pulled from omega-dot glass (o.d. 1-2 mm) on a Brown Flaming horizontal puller and were usually filled with 3M-KCl or 3M-KMeSO4. When it was necessary to make large changes in the membrane potential the electrodes were filled with 3M-CsCl to block potassium currents. When cells were filled with bis (O-aminophenoxy) ethane-n,n,n',n'- tetraacetic acid (BAPTA), K4BAPTA was dissolved in 3M-KCl to give a concentration of 2 mm. This solution was used to fill the tip of the microelectrode and the electrode was then backfilled with KCl as usual. Signals were amplified using a M-77 (WP Instruments) amplifier or an Axo-Clamp (Axon Instruments) and stored on an IBM PC-AT compatible microcomputer using a modified version of P-Clamp (Axon Instruments). Records were analysed off-line using a modified version of P-Clamp. In voltage-clamp experiments the headstage of the Axo-Clamp was continuously monitored and a switching frequency of 2-4 khz was used. Bipolar steel stimulating electrodes (Frederick Haer) were inserted into the corpus callosum. Afferent fibres were stimulated using an isolated stimulating unit. During LTP experiments, stimuli were delivered at 1 Hz. The stimulus

3 POTENTIATION OF CORTICAL SYNAPSES protocol used to elicit LTP was two tetani at 1 Hz for 1 s separated by 2 s at twice the stimulus intensity. In order to minimize contamination from voltage-dependent conductances and polysynaptic inputs the initial slope of the EPSP was used as the measure of synaptic efficacy. To average data for LTP experiments across cells, the data from all the cells to be averaged were aligned with the time of the tetanus (or pairing) being assigned as time zero. The data were then binned into either 5 or 1 min bins. All points in each bin were then averaged. Each data point is shown as mean+s.e.m. All bath applied drugs used in this study were dissolved directly in the bathing medium. Drugs used were DL-2 amino-5-phosphonovalerate (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Cambridge Research Biochemical), BAPTA (Molecular Probes), and Picrotoxin (Sigma). 617 RESULTS Stable intracellular recordings were obtained from eighty-six pyramidal neurones in layer V and layer VI. In cells which were impaled with KCl- or KMeSO4-filled microelectrodes the average resting membrane potential was mv (n = 25). Stimulation in the corpus callosum led to the generation of an EPSP. With small stimulus strengths this EPSP was graded in amplitude, had a fixed latency and monophasic decay (Fig. IA). As the stimulus strength was increased, however, a later slow polysynaptic component was apparent (Fig. IA). This polysynaptic component was more easily generated in the presence of picrotoxin, but, even without picrotoxin, it invariably appeared as the stimulus strength was increased. Note, however, that the early part of the EPSP is smoothly graded with stimulus intensity, suggesting that it is monosynaptic in nature (Fig. lab). In order to discriminate between monosynaptic and polysynaptic EPSPs, two experiments were carried out. Firstly, raising the concentration of divalent ions in the extracellular medium (12 mm-ca2+, 12 mm-mg2") has been shown to suppress polysynaptic EPSPs (Jahr & Jessel, 1985; Jahr & Yoshioka, 1986), presumably by increasing the threshold for spike generation in interneurones (Frankenhaeuser & Hodgkin, 1957). As shown in Fig. 1B, raising the divalent ion concentration abolished the late components of the EPSP, leaving a monophasic EPSP with a fixed latency. In the second experiment the afferents were repetitively stimulated at frequencies of 1-3 Hz. The late components of the EPSP were unable to follow the stimulus at these frequencies (data not shown). These data indicate that if analysis is restricted to the initial rising phase of the EPSP, one can be reasonably confident that a monosynaptic EPSP is being studied. Although glutamate and/or aspartate is generally thought to be the transmitter mediating fast EPSPs in the cortex, the identity of the transmitter released by stimulation of the callosal inputs is not known. Binding studies indicate a high concentration of L-glutamate binding sites in the anterior cingulate cortex (Monaghan & Cotman, 1985; Cotman, Monaghan, Ottersen & Storm-Mathisen, 1987). At the synapse between the Schaffer collateral-commissural fibre and the CAl pyramidal cells where glutamate is the presumed transmitter, synaptically released glutamate has been shown to activate both the N-methyl-D-aspartate (NMDA) and non-nmda class of receptor (Collingridge, Herron & Lester, 1988 a; Kauer, Malenka & Nicoll, 1988b; Andreasen, Lambert & Jensen, 1989; Hestrin, Nicoll, Perkel & Sah, 199). We therefore examined the pharmacological profile of the EPSP in the cingulate. The effects of the selective non-nmda antagonist CNQX (Blake, Brown

4 618 P. SAH AND R. A. NICOLL A a B a b 15 - cn1 b 1 mv b b ~~~~~~~~~~~~~~2 ms -~~~~ L Stimulus (% of threshold) Fig. 1. The excitatory input from the corpus callosum to layer V and layer VI pyramidal neurones in anterior cingulate cortex contains a monosynaptic component. Aa, EPSPs recorded in a layer V and layer VI pyramidal neurone in response to stimuli of increasing intensity in the corpus callosum. Ab, the peak of the EPSP is plotted as a function of stimulus strength. Ba, EPSPs recorded in another neurone at a high stimulus intensity in normal Ringer solution (2 mm-calcium, 2 mm-magnesium). A polysynaptic component can be clearly seen in the EPSP which is abolished when Ringer solution containing 12 mm- Ca2+ and 12 mm-mg2+ is applied, leaving a fixed latency monosynaptic input (Bb). In each case six successive EPSPs have been superimposed. -8 mv -5 mv CNQX \Control CNQX CNQX + APV 5 mv 2 ms Fig. 2. The excitatory input from the corpus callosum to layer V and layer VI pyramidal neurones activates both NMDA and non-nmda receptors. At a negative membrane potential (-8 mv), stimulation of the afferent fibres generates an EPSP which is largely abolished by the non-nmda antagonist CNQX (1 sm, left records). Depolarization of the postsynaptic cell to -5 mv by current injection reveals a slower EPSP which is entirely blocked by the NMDA antagonist DL-APV (5 #M). & Collingridge 1988; Honore, Davies, Drejer, Fletcher, Jacobsen, Lodge & Nielsen, 1988) are illustrated in Fig. 2. At the resting membrane potential, -8 mv, 1 /M- CNQX, almost abolishes the EPSP (n = 9). In the presence of CNQX, depolarization of the postsynaptic cell to -5 mv, reveals an EPSP with slower kinetics which is

5 POTENTIATION OF CORTICAL SYNAPSES completely blocked by the NMDA receptor antagonist APV (5 1uM, n = 5). Thus, as with the hippocampus, activation of afferents onto the layer V and layer VI pyramidal cells also activates both NMDA and non-nmda type receptors. Apart from the EPSP described so far, we noticed that during a tetanus delivered at a high enough stimulus intensity, and even with single strong stimuli, a slow 619 A a 1.2T 1-23T 1*27T 1*47 T J..J J W~~~~~~~~~~~~2 mv I ~~~~~2 ms b 1.27T l*47t.5 T 1.7T U ~~~J 1 s B C D 1~~~~ 15 Fast EPSP -8 mv 1 X Slow EPSP E 5 +3mV +1mV (I) - Wi L A"A Stimulus (% of threshold) 1 s 1 s j.5na Fig. 3. A slow EPSP accompanies the polysynaptic burst elicited by high stimulus strengths. The top records in Aa show the fast EPSP evoked by increasing stimulus strengths which are expressed in terms of the threshold stimulus (T) required to evoke an EPSP. The bottom records in Ab show the responses at a slower time base. B plots the records partially shown in A. The amplitude of the slow EPSP was measured at 1 s after the stimulus. C shows the slow EPSP at -8 and at + 3 mv membrane potential. D shows voltage clamp records from another cell at -8 and + 1 mv. depolarizing potential was apparent (n = 5). The slow EPSP was an all-or-nothing event and coincided with the all-or-nothing production of synchronous polysynaptic EPSPs. As illustrated in Fig. 3A, as the stimulus intensity is increased the fast EPSP increases in amplitude (as more afferent fibres are recruited (Fig. 3Aa)), and at a sufficiently high stimulus, a synchronous polysynaptic EPSP is generated. Following the polysynaptic EPSP, a slow depolarizing potential is apparent, which does not increase further in amplitude (Fig. 3Ab). The amplitude of the fast EPSP, and the amplitude of the slow depolarizing potential (measured at 1 s after the stimulus) are

6 62 P. SAH AND R. A. NICOLL shown in Fig. 3B as functions of stimulus intensity. It was possible to reverse the slow potential by depolarizing the postsynaptic cell (Fig. 3C) and it had a reversal potential of around mv. Under voltage clamp at negative membrane potentials an inward current was measured, which could also be reversed at positive potentials A o % 1 2 B - C/) a- w Tetanus Time (min) 4 ms 2 mv 1 2 Fig. 4. Long-term potentiation at the excitatory synapse between callosal afferents and layer V and layer VI pyramidal neurones. A, the initial slope of the EPSP is plotted as a function of time for twelve neurones. At time zero, a brief tetanus was delivered as described in the methods. B, sample records taken from one cell before and after the tetanus (1 and 2 respectively in A). The two traces are superimposed at the right and show the marked increase in the initial slope following the tetanus. (Fig. 3D). Furthermore, the slow potential could not be reproduced by direct injection of current into the postsynaptic cell. These observations suggest that the slow potential is synaptic in nature. Tetanic stimulation of the callosal afferents led to a long lasting increase in the EPSP. Out of sixty-four cells in which tetanic stimulation was delivered, fifty-five cells showed an increase in the slope of the EPSP for at least 1 min and nine cells showed no change in the EPSP. Figure 4, shows the average data from twelve neurones in which the EPSP was monitored for a period of 1 min before the delivery of a brief tetanic stimulus. Following the tetanus, the slope of the EPSP showed an immediate increase which then slowly declined over the next 6 min. Sample records from one cell are shown in Fig. 4B. There was no change in resting membrane properties of the cell after the tetanus. While in most experiments the potentiation was decremental, for simplicity we will refer to it as LTP. In the visual cortex, it has

7 POTENTIATION OF CORTICAL SYNAPSES been reported that it was necessary to block inhibition in order to elicit LTP (Artola & Singer, 1987). In the cingulate, however, LTP could be obtained routinely without addition of picrotoxin to the bathing Ringer solution, as has been reported in sensorimotor cortex by Baranyi & Szente (1987) and Sutor & Hablitz (1989a). 621 A 3 - a- 2 1 B w +~4m Tetanus zioo 2 Time (min) AL 2~~~1m Fig. 5. Long-term potentiation is blocked in the presence of DL-APV. A, the initial slope of the EPSP is plotted as a function of time for five neurones. At time zero, a brief tetanus was delivered as described in the methods. B, sample records taken from one cell before and after the tetanus (1 and 2 respectively in A). The two traces are superimposed at the right and show that in the presence of APV (5,uM), LTP is blocked. In the CAl region of the hippocampus, it has been demonstrated that although normal synaptic transmission uses the non-nmda class of receptor, activation of NMDA receptors is essential for eliciting LTP (Collingridge, Kehl & McLennan, 1983). As shown above, the callosal input to cingulate layer V and layer VI cells activates both the non-nmda and the NMDA class of glutamate receptor. We therefore tested the effects of APV on the LTP. DL-APV (5 /tm) had small, variable depressant effects on the EPSP. The induction of LTP was, however, completely blocked by addition of APV to the extracellular solution, as shown in Fig. 5 (data from five cells). Following wash-out of the APV, normal LTP could be produced in the same cell (n = 2). It has been found that buffering postsynaptic calcium prevents LTP in the CA1 region of the hippocampus (Lynch, Larson, Kelso, Barrionuevo, & Schottler 1983; Malenka, Kauer, Zucker & Nicoll, 1988), and in neocortex (Baryani & Szente, 1987). To test if a rise in postsynaptic calcium is also necessary for the induction of LTP in the cingulate cortex, cells were loaded with the calcium buffer BAPTA. After impalement, the cells were allowed to stabilize for 2 min to allow the

8 622 P. SAH AND R. A. NICOLL cell to fill with BAPTA. Baseline responses were then obtained for a further 1 min before giving the tetanus. As shown in Fig. 6, (n = 7) cells loaded with BAPTA, did not show LTP. Two further predictions can be made based on the findings outlined above. Firstly, it should be possible to demonstrate the phenomenon of co-operativity (McNaughton, A 3 c 2 Nol 1 + B L Tetanus Time (min) J 1 mv 4 ms Fig. 6. Long-term potentiation is blocked by intracellular BAPTA. Electrodes were filled with KBAPTA as described in the methods. A, the initial slope of the EPSP is plotted as a function of time for seven neurones. At time zero, a brief tetanus was delivered as described in the methods. B, sample records taken form one cell before and after the tetanus (1 and 2 respectively in A). The two traces are superimposed at the right and show that in cells filled with BAPTA, LTP is blocked. Douglas & Goddard, 1978; Lee, 1983). That is, it should be possible to find a stimulus intensity at which a tetanus delivered to the afferent fibres is inadequate to elicit LTP. When the stimulus strength is subsequently increased normal LTP should be elicited. Secondly, pairing postsynaptic depolarization with low frequency stimulation of the inputs should lead to LTP (Wigstrdm, Gustaffson, Huang & Abraham, 1986; Gustaffson, Wigstr6m, Abraham & Huang, 1987). These two predictions are tested in Fig. 7. In Figure 7A the idea of co-operativity was tested. It can be seen that in this cell the weak tetanus had no effect on the EPSP slope whereas the strong tetanus (stimulus strength 2 x weak stimulus) did give LTP. Similar co-operativity was seen in nine other cells. The effect of pairing low frequency synaptic activation with postsynaptic depolarization is shown Fig. 5B. In the CAl region of the hippocampus, pairing ten to twenty EPSPs with depolarization at 41 Hz is adequate

9 POTENTIATION OF CORTICAL SYNAPSES to produce robust LTP (Kauer et al b). In the cingulate we found this protocol to have little effect on the slope of the EPSP. In fact in eight cells, it proved impossible to produce an increase in the EPSP with low frequency stimulation with up to forty stimuli. In three of these cells subsequent tetanization produced 623 A 3 - C o2 1: 1 Weak Strong B d 2 - ) - 1 L Pair Tetanus Time (min) Fig. 7. Long-term potentiation in the cingulate shows co-operativity but does not occur with pairing EPSPs with postsynaptic depolarization. A, the initial slope of the EPSP is plotted for one cell against time. At the time labelled 'weak', a weak tetanus (1 Hz, 1 s repeated twice, 2 s apart at the control stimulus strength) was delivered. There is no change in the EPSP slope. At the time labelled 'strong' the same tetanus was again delivered but the stimulus strength was doubled during the tetanus. There is a rapid and sustained increase in the slope of the EPSP. B, the initial slope of the EPSP is plotted for a different cell against time. During the time labelled 'pair' the membrane of the postsynaptic cell was depolarized to around the reversal potential of the EPSP and the afferents were stimulated 4 times at 1 Hz. The membrane potential was then returned to the resting potential. There is no change in the slope of the EPSP. At the time labelled 'tetanus' a strong tetanus was delivered which resulted in a rapid and sustained increase in the slope of the EPSP. potentiation indicating that these synapses could be potentiated. The data for one of these cells are illustrated in Fig. 7B. During the time labelled 'pair' the postsynaptic cell was depolarized to the reversal potential (- mv) of the EPSP and

10 624 P. SAH AND R. A. NICOLL the afferent fibres stimulated at 1 Hz to pair the EPSPs with depolarization forty times. It can be seen that this manipulation had no effect on the initial slope of the EPSP. Subsequently the afferents were tetanized as described before and clear LTP was produced. A -5 3 o a k en t t t n Weak tetanus Weak tetanus + depol Strong tetanus B LU Time (min) 4 ms 2 mv Fig. 8. Pairing of a weak tetanus with postsynaptic depolarization does not lead to LTP. A, the initial slope of the EPSP is plotted against time for six cells. The three panels are continuous in time, spanning a total of 9 min. In the left panel it is shown as in Fig. 6 that a weak tetanus does not lead to LTP. In the centre panel, at time zero, the cell is depolarized (depol) to the reversal potential of the EPSP and the same weak tetanus again delivered. It can be seen that there is no change in the slope of the EPSP. Subsequently a strong tetanus is delivered to the same cell and LTP is demonstrated. Sample traces from one cell are superimposed before and after the weak tetanus (left panel, 1 and 2 in A), before and after the pairing (middle panel, 3 and 4 in A), and before and after the strong tetanus (right panel, 5 and 6 in A). The experiments thus far demonstrate that, as for the CAI region of the hippocampus, tetanization of the afferents leads to LTP which is blocked by the NMDA antagonist APV and also by buffering the postsynaptic calcium using BAPTA. These data are consistent with the hypothesis that LTP is triggered by an influx of extracellular calcium through NMDA receptor operated channels, which are unblocked during the depolarization resulting from the tetanus. One clear exception, however, is the failure to elicit potentiation by pairing low frequency afferent stimulation with postsynaptic depolarization. It is known that the density of NMDA binding sites in the cingulate cortex is lower than that in the CAl region of the hippocampus (Monaghan & Cotman, 1985). In addition the NMDA component of EPSPs has been demonstrated to show a marked summation (Collingridge, Herron & Lester, 1988 b). Thus, it is possible that insufficient NMDA receptor activation occurred during low frequency pairing. In an

11 POTENTIATION OF CORTICAL SYNAPSES attempt to control for this we paired postsynaptic depolarization with a tetanus that contained the same number of stimuli as used to evoke LTP but the stimulus strength was below threshold for evoking LTP. Figure 8 shows averaged results from six experiments in which this idea was tested. Cells were impaled and after the baseline was obtained, a weak tetanus which did not elicit LTP was delivered. Following the tetanus, another baseline was obtained and the cell was depolarized to around the reversal potential of the EPSP (- mv). The weak tetanus was again delivered. As can be seen there was no increase in the EPSP. Following this a strong tetanus was delivered which produced normal LTP indicating that the synapses in these experiments were capable of generating LTP. This inability to produce LTP, by pairing the EPSP with postsynaptic depolarization, clearly differs from that obtained in the CAI region of the hippocampus. 625 DISCUSSION The results described in this study confirm and extend the findings of Vogt & Gorman (1982) that there is a monosynaptic, excitatory input from the corpus callosum onto layer V and layer VI pyramidal neurones in the anterior cingulate cortex. The transmitter released by this input activates both NMDA and non-nmda glutamate receptor subtypes. Brief tetanic stimulation of these afferents leads to a long-lasting increase in the EPSP. The induction of LTP shows co-operativity and can be blocked by APV in the extracellular medium or by filling the postsynaptic cell with the calcium buffer BAPTA. These properties are identical to those of the Schaffer collateral-commissural synapse onto CAI pyramidal cells in the hippocampus. However, it was not possible to produce LTP by applying weak tetani with concomitant postsynaptic depolarization, a manipulation which produces robust LTP in CAl pyramidal neurones. It has been previously shown that the layer V and layer VI pyramidal neurones receive an excitatory input from the pyramidal cells in the contralateral cingulate via the corpus callosum. This input is preserved in a coronal slice from the cingulate cortex (Vogt & Gorman, 1982). Since the interpretation of many of the experiments on the mechanisms underlying LTP require that the synaptic potential is monosynaptic we went to considerable lengths to ensure that the EPSPs analysed in our study were monosynaptic. Thus a number of electrophysiological criteria were used to establish that the initial component of the EPSP elicited by stimulating the corpus callosum was monosynaptic. (1) It has a fixed latency. (2) As the stimulus intensity is increased, the peak EPSP is smoothly graded with the stimulus intensity. (3) When the concentration of divalent cations in the extracellular medium is increased, the polysynaptic activity is suppressed but the monosynaptic component remains at the same fixed latency. (4) The early component can follow a high frequency train at up to 3 Hz. (5) In the presence of CNQX an NMDA synaptic component of similar latency was recorded. In the mammalian central nervous system glutamate binds to three types of receptors distinguished by their selective ligands NMDA, quisqualate and kainate (Watkins & Evans, 1981). The quisqualate and kainate receptors are lumped together as non-nmda receptors because of the difficulty of separating them

12 626 P. SAH AND R. A. NICOLL pharmacologically. In the hippocampus, synaptically released glutamate activates both the NMDA and non-nmda subtypes of receptor (Collingridge et al. 1988a; Kauer et al. 1988b; Andreasen et al. 1989; Hestrin et al. 199). However, because the NMDA channel is normally blocked in a voltage-sensitive manner by extracellular Mg2" (Nowak, Bregestovski, Ascher, Herbet & Prochiantz, 1984; Mayer, Westbrook & Guthrie, 1984) little NMDA receptor mediated EPSP is seen at resting membrane potentials. Depolarization of the postsynaptic cell relieves the block of the NMDA channel and the EPSP mediated by this receptor, which has a much slower time course than the non-nmda EPSP, becomes apparent. The EPSP examined in this study was found to have pharmacological properties very similar to those in the CAI region of the hippocampus. Thus, at negative membrane potentials, the EPSP was essentially blocked by the selective non-nmda antagonist CNQX. Depolarization of the postsynaptic cell revealed a slower EPSP which was blocked by the NMDA receptor antagonist APV. These observations strongly suggest that the transmitter mediating the EPSP at this synapse is also glutamate. At high enough stimulus strengths, a slow depolarizing potential was seen. This potential was blocked by CNQX and could not be reproduced by direct current injection into the cell suggesting that it is synaptic in nature. Slow EPSPs which have been previously described in the CNS have been mediated by a closure of K+ channels (see Nicoll, 1988.) The slow EPSP described here could not be blocked by intracellular caesium and had a reversal potential close to mv which means that it is not mediated by K+ ions. We were unable to block the slow potential by addition of propranalol (3 /am), atropine (3/M), cimetidine (3 /tm), spiperone (3 /tm), or APV (5 1aM; not illustrated). Many neurones throughout the nervous system have been shown to contain neuropeptides and in general the responses to peptides have a slow time course (Somogyi, Hodgson, Smith, Nunzi, Gorio & Wu, 1984; Sloviter & Nilaver, 1987; Iversen, 1984; H6kfelt, Lundberg, Schultzberg, Johansson, Skirboll, Anggard, Fredholm, Hamberger, Pernow, Rehfeld & Goldstein, 198). Thus it is possible that a peptide might mediate this potential. Tetanic stimulation of the afferents led to a long lasting increase in the size of the EPSP. As for the Schaffer collateral-commissual to CAI synapse, the LTP reported here displayed co-operativity and was blocked by the NMDA receptor antagonist APV, or by buffering the calcium in the postsynaptic cell with BAPTA. A similar sensitivity to APV has been reported for LTP in layer II-JV of visual cortex (Artola & Singer, 1987), and in frontal cortex (Sutor & Hablitz, 1989b). In motor cortex it has been shown that intracellular EGTA blocks LTP (Baranyi & Szente, 1987). These observations suggest that, as for the CAI region of the hippocampus, activation of NMDA receptors and a rise in postsynaptic calcium ions are necessary to elicit LTP in cortical neurones. It is notable that the LTP that we report typically declined over 4-5 min following the tetanus. LTP in CAl pyramidal cells is stable from many hours (Bliss & Lynch, 1988). Non-decremental LTP has also been reported in neocortex (e.g. Baranyi & Szente, 1987; Sutor & Hablitz, 1989a). The basis for this difference in time course is unknown. One clear problem with the hypothesis developed in CAI is that pairing a weak tetanus with depolarization failed to produce LTP. There are several possibilities to explain this discrepancy. Firstly, it may be that the electrotonic structure of the

13 POTENTIATION OF CORTICAL SYNAPSES neurone is such that current injected at the soma is not effectively 'seen' by the stimulated synapse. We think this possibility is unlikely because it was possible to reverse the EPSP by current injection demonstrating that current injected at the soma is seen by the subsynaptic membrane. Secondly, it is possible that conjunction of presynaptic activity with depolarization of the postsynaptic cell is either not necessary or sufficient to generate LTP. In this scenario the co-operativity that is seen here is not due to changes in membrane potential but some other interaction between synapses. For instance, the concentration of glutamate at any one activated excitatory synapse may be increased by activation of neighbouring synapses, as has been suggested for inhibitory synapses on the Mauthner cell (Faber & Korn, 1988). Thirdly, the present results establish that NMDA receptors are monosynaptically activated on the impaled neurone. However, it is possible that NMDA receptors on interneurones are required for the induction of LTP. These receptors would be uninfluenced by depolarization of the impaled neurone. During a strong tetanus, it is clear that polysynaptic pathways are activated, at least during the early part of a tetanus. It is possible that NMDA receptors are involved at one of these synapses and blockade of these receptors prevents activation of a local circuit which is essential for the generation of LTP. Activation of interneurones during the strong tetanus could release a factor associated with the slow EPSP that is essential for the generation of LTP. We have noticed a strong correlation between the production of the slow EPSP-and the ability to elicit LTP, suggesting that the two phenomena may be related. This putative factor could act at the level of the NMDA receptor/channel or it could act at a stage after the entry of calcium. Transmitters such as glycine (Johnson & Ascher, 1987; Thomson, 1989) and serotonin (Nedergaard, Engberg & Flatman, 1987; Reynolds, Baskys & Carlen, 1988), which have been shown to enhance NMDA responses are unlikely to be the released substance since pairing in the presence of these substances still failed to elicit potentiation. In addition we tested vasoactive intestinal polypeptide (1 ftm), somatostatin (1 /SM), vasopressin (1 JtM), substance P (1 /tm) and cholecystokinin (1 /tm) with pairing and failed to get potentiation. Alternatively, although activation of NMDA receptors does lead to a rise in postsynaptic calcium a second factor coupled to second messenger systems could be required for the production of LTP. In this regard it is interesting that in the CAI region, attempts to generate LTP by ionophoresis of NMDA were unsuccessful, raising the possibility of a second 'cofactor' (Kauer et al. 1988b). It has been demonstrated that pairing EPSPs with postsynaptic depolarization 25-5 times can produce LTP in a fraction of cells in layer V and layer VI of sensorimotor and motor cortex (Baranyi & Szente, 1987; Bindman, Murphy & Pockett, 1988). This raises the possibility that differences might exist in cortical LTP. In conclusion, this study shows that the neurotransmitter at the excitatory synapse between callosal afferents and the layer V and layer VI pyramidal cells in anterior cingulate cortex activates both NMDA and non-nmda receptors. Brief repetitive stimulation of the pathway produces a form of LTP which shares many but not all the properties of LTP at the Schaffer collateral-commissural synapse in the CAl region of the hippocampus. 627

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