application of SQ22,536 slightly increased the amplitude of the (a.h.p.) and reduced

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1 J. Phy8iol. (1986), 372, pp With 8 text-figures Printed in Great Britain CYCLIC ADENOSINE 3', 5'-MONOPHOSPHATE MEDIATES fl-receptor ACTIONS OF NORADRENALINE IN RAT HIPPOCAMPAL PYRAMIDAL CELLS BY D. V. MADISON AND R. A. NICOLL From the Departments of Pharmacology and Physiology, University of California, San Francisco, CA 94143, U.S.A. (Received 13 February 1985) SUMMARY 1. Intracellular recordings were made from rat hippocampal CAI pyramidal neurones in the in vitro slice preparation to study the actions of cyclic adenosine 3',5'-monophosphate (cyclic AMP). 2. Application of the membrane permeant analogue of cyclic AMP, 8-Br cyclic AMP caused a small depolarization of the resting membrane potential accompanied by an increase in membrane input resistance and also reduced the amplitude of depolarization-evoked calcium-activated potassium after-hyperpolarizations (a.h.p.s) Br cyclic AMP reduced calcium-activated a.h.p.s but did not reduce calcium action potentials in these cells. 8-Br cyclic AMP also reduced action potential frequency accommodation. 4. The effects of 8-Br cyclic AMP were not mimicked by cyclic AMP applied extracellularly but were imitated by intracellular injections of cyclic AMP. 5. Activation of the endogenous adenylate cyclase of pyramidal cells either by intracellular injection of the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate, or by extracellular application of forskolin, reduced the a.h.p. and accommodation. 6. Reducing phosphodiesterase activity with application of either 3-isobutyl- 1-methylxanthine or Ro reduced the amplitude of the a.h.p. and potentiated the a.h.p.-blocking action of noradrenaline. Reducing adenylate cyclase activity by application of SQ22,536 slightly increased the amplitude of the (a.h.p.) and reduced the a.h.p.-blocking action of noradrenaline. 7. We conclude that the fl-receptor actions of NA on hippocampal CAI pyramidal cells are mediated by intracellularly produced cyclic AMP. INTRODUCTION Since its characterization as a second messenger (cf. Rall & Sutherland, 1961; Sutherland, 0ye & Butcher, 1965), many studies have sought to determine the role that cyclic adenosine 3',5'-monophosphate (cyclic AMP) plays in neurotransmission, particularly noradrenergic transmission (cf. Drummond, 1983, for review). In the

2 246 D. V. MADISON AND R. A. NICOLL cerebellum and cerebral cortex, it has been reported that incubation of tissue slices with noradrenaline (NA) causes a two to twentyfold increase in tissue levels of cyclic AMP over basal levels (Kakiuchi & Rall, 1968; Kakiuchi, Rall & McIlwain, 1969; Rall & Sattin, 1970; cf. Bloom, 1975). In the hippocampus, NA has been reported to produce substantial increases in tissue cyclic AMP levels (Palmer, Sulser & Robison, 1973). This NA-induced rise in hippocampal cyclic AMP levels has been reported to occur primarily in neuronal cells (Segal, Greenberger & Hofstein, 1981) and to be due to activation of #,/-receptors (Dolphin, Hamont & Bockaert, 1979). Application of exogenous cyclic AMP has been reported in several studies to mimic fl-receptormediated actions of NA in the cerebellum (Siggins, Hoffer & Bloom, 1971 a; Siggins, Oliver, Hoffer & Bloom, 1971 b; Hoffer, Siggins, Oliver & Bloom, 1972), and in the hippocampus (Segal & Bloom, 1974; Segal, 1981), although these actions are somewhat different from those described here. In this paper, we report on evidence that the f-receptor-mediated actions of NA in the hippocampus, as described in the accompanying report (Madison & Nicoll, 1986), are mediated through the intracellular second messenger cyclic AMP. Some of these findings have appeared in a preliminary report (Madison & Nicoll, 1982). METHODS The methods employed in this report are similar to those described in the accompanying report (Madison & Nicoll, 1986). Experiments were performed in the rat hippocampal slice preparation using intracellular recording from CAI pyramidal neurones to study the actions of cyclic AMP. Drugs applied via the superfusing medium were: 8-bromoadenosine 3',5'-monophosphate (8-Br cyclic AMP, sodium salt), (-)isoprenaline HCl, propranolol HCl, (-)noradrenaline HC1 or bitartrate, tetraethylammonium chloride (TEA), tetrodotoxin (TTX), adenosine chloride, cyclic AMP, and 3-isobutyl-I-methyl xanthine (IBMX), which were obtained from Sigma. Forskolin was obtained from Calbiochem. Ro was a gift from Hoffman-La Roche and SQ22,536 (9- (tetrahydro-2-furyl) adenine) was a gift from Squibb. Some drugs were applied intracellularly by injecting them through the recording electrode. These were cyclic AMP, adenosine 5'-monophosphate (5' AMP, Sigma) and 5'-guanylyl-imidodiphosphate (Sigma), all prepared in 2 M-potassium methylsulphate (KMeSO4) at a concentration of 100 mm. These compounds were allowed to leak from the electrode without any driving current. In one set of experiments, isoprenaline (1 M) was applied to the preparation by ionophoresis. For measurement of the after-hyperpolarization (a.h.p.) amplitude in standard medium, the peak hyperpolarizing voltage of the slow a.h.p. component was taken relative to resting potential. For a.h.p.s recorded in TTX and TEA where a single phase of hyperpolarizing response was most often seen, the amplitude of this single hyperpolarization was measured. In all experiments in this report, recording electrodes were filled with potassium methylsulphate (2 M). RESULTS Mimicry of noradrenergic fl-receptor actions by cyclic AMP Application of the membrane permeant analogue of cyclic AMP, 8-Br cyclic AMP, mimics the fl-receptor-mediated actions of NA on hippocampal CAl pyramidal cells. These noradrenergic actions, as reported in the accompanying paper (Madison & Nicoll, 1986), are a depolarization of the resting membrane potential of these neurones accompanied by an increase in the membrane input resistance, and a blockade of action potential-evoked calcium-activated potassium a.h.p.s. As shown in Fig. 1, a brief ionophoretic application of the,8-receptor agonist, isoprenaline (100 na for 2 s),

3 CYCLIC AMP AND NORADRENALINE IN HIPPOCAMPUS 247 Isoprenaline Isoprenaline 8-Br cyclic AMP i + A propranolol propranolol V ; "011 B Control Br cyclic AMP 4 min Io/-< 51glD 'mv 2 5 min -d.c. Fig Br cyclic AMP mimics the fl-receptor-mediated actions of NA in hippocampal CAl pyramidal cells. A, chart records from a pyramidal cell bathed in TTX (1 /SM) and TEA (5 mm). Upward deflexions are calcium action potentials evoked by depolarizing current pulses and downward deflexions are the resulting a.h.p.s. The action potential amplitude in this and other chart records is truncated by the chart recorder. lonophoretic application of isoprenaline (100 na for 2 s, applied at arrow) caused a depolarization of the membrane potential and a reduction in the amplitude of the a.h.p.s recorded simultaneously (AI). Addition of propranolol (10 jum) to the bathing medium blocked the action of isoprenaline (A2), but application of 8-Br cyclic AMP (bath application, 1 mm, with propranolol still in medium) caused a depolarization and a decrease in a.h.p. amplitude (A3). The membrane potential of this cell was -64 mv. B, chart records from another pyramidal cell bathed in normal medium. Bi, the a.h.p. which follows a short duration (approx. 60ms) depolarizing current stimulus. B2, downward deflexions are constant-current hyperpolarizing pulses. Application of 8-Br cyclic AMP (1 mm) caused a depolarization of the membrane potential and an increase in membrane input resistance. The membrane was repolarized to control membrane potential with steady hyperpolarizing current at the point marked by the bar (-d.c.) to show the change in resistance more clearly. B3, the slow component of the a.h.p. evoked by depolarizing current was reduced by 8-Br cyclic AMP application. The membrane potential of this cell was -56 mv. like NA, caused a slow, long-lasting depolarization of the resting membrane potential and a reduction in the amplitude of the a.h.p. (Fig. 1 Al). Bath application of propranolol (10 /M) attenuated these responses to isoprenaline (Fig. 1 A2), but application of 8-Br cyclic AMP (1 mm in the bathing medium), caused effects similar to isoprenaline, as it depolarized the cell and reduced the a.h.p. (Fig. 1 A3). Since these actions of 8-Br cyclic AMP occurred even when the f-receptor was blocked with propranolol, this suggested that the analogue was bypassing the f-receptor to exert its effects. As with noradrenergic f-receptor activation, the depolarization caused by application of 8-Br cyclic AMP was accompanied by an increase in membrane input resistance as measured with brief hyperpolarizing current pulses passed through the

4 248 D. V. MADISON AND R. A. NICOLL A 1 Control 8-Br cyclic AMP Wash 2 I 25 mv 0-25 na 50 mns 8 J 2 s 10 mv Fig Br cyclic AMP reduces calcium-activated potassium a.h.p.s without reducing calcium action potentials in CAI pyramidal cells. All records shown are from the same pyramidal cell bathed in TTX (1 SUM) and TEA (5 mm). A, digital plotter records of calcium action potentials evoked by a brief depolarizing current stimulus passed through the recording electrode. The current monitor trace is below the voltage trace. A2, superimposed traces of control record and record obtained in 8-Br cyclic AMP. B, chart records of the a.h.p. that followed the action potentials shown in AI. Application of 8-Br cyclic AMP (1 mm) markedly reduced the a.h.p., but did not reduce the corresponding calcium action potential. The membrane potential of this cell was -62 mv. recording electrode (Fig. 1 B2). Note also that the a.h.p. recorded during application of 8-Br cyclic AMP was reduced compared to control levels (n = 15) (Fig. 1 B3). The mechanism by which 8-Br cyclic AMP reduces the calcium-activated potassium a.h.p. appears to be identical to that of noradrenergic a.h.p. blockade. NA reduces a.h.p.s without reducing calcium entry into the pyramidal cell (Madison & Nicoll, 1986). Calcium action potentials were evoked in pyramidal cells bathed in TTX (1,LM) and TEA (5 mm) (Fig. 2A). Each such action potential in turn produced a calciumactivated potassium a.h.p. (Fig. 2B). While application of 8-Br cyclic AMP markedly reduced the amplitude of the a.h.p., it had very little effect on the preceding calcium action potential (n = 5). Thus, 8-Br cyclic AMP, like NA, appears to reduce calcium-activated potassium a.h.p.s without reducing calcium entry. Injection of cyclic AMP into the cell through the recording electrode (100 mm) also abolished the a.h.p. (see below), but in cells recorded in TTX and TEA, little change in the amplitude or duration of the calcium action potential was observed (not shown). In some of these cells, the base of the calcium action potential was slightly prolonged. 8-Br cyclic AMP, like NA, reduced accommodation of pyramidal cell action potential discharge through antagonism ofcalcium-activated potassium conductance. Pyramidal cells, bathed in normal medium, responded to long duration depolarizing current pulses with a train of action potentials that accommodated during the course of the discharge (Fig. 3A; Madison & Nicoll, 1982, 1984; Lanthorn, Storm & Andersen, 1984). This discharge was associated with an a.h.p. which could be seen more clearly following a short (70 ms) duration depolarizing pulse (Fig. 3B). Application of 8-Br cyclic AMP (1 mm) reduced the a.h.p. (Fig. 3B) and the accommodation of action potential discharge such that the cell fired throughout the

5 CYCLIC AMP AND NORADRENALINE IN HIPPOCAMPUS 249 A Control 8-Br cyclic AMP Wash B I5 mnv i s Fig Br cyclic AMP reduces accommodation in hippocampal CAl pyramidal cells. All records taken from the same pyramidal cell bathed in normal medium. A, photographic records of the response of a pyramidal cell to a long (700 ms) depolarizing current pulse which elicits an accommodating train of action potentials. B, chart records of the response of the cell to a short duration (70 ms) depolarizing current stimulus which elicits an a.h.p. Bath application of 8-Br cyclic AMP (1 mm for 10 min) caused a reduction in the slow component of the a.h.p. and in accommodation pari passt. The effects of 8-Br cyclic AMP were reversed 20 min after removing the drug from the superfusate. The membrane potential of this cell was -61 mv. long current pulse, and the frequency of the initial discharge was increased (Fig. 3 A, n = 5). These effects were reversible upon washing the cyclic AMP analogue from the recording chamber. In the presence of 8-Br cyclic AMP (1 mm), these pyramidal cells fired action potentials in response to a strong 600 ms long current stimulus as opposed to P5 in control conditions. Site of cyclic AMP action One possibility that could account for the actions of 8-Br cyclic AMP on pyramidal neurones is that this cyclic AMP analogue might be exerting its actions by binding to extracellular adenosine receptors, rather than by promoting cyclic AMP-dependent intracellular processes. To control for such a possibility, adenosine and cyclic AMP were applied to pyramidal cells. The action of adenosine (10 gsm) did not resemble that of 8-Br cyclic AMP in that it caused a hyperpolarization of the membrane potential (see also Segal, 1981) and did not cause a reduction in the a.h.p. (Fig. 4A, n = 5). Higher concentrations produced a larger hyperpolarization associated with a clear decrease in input resistance. Cyclic AMP (100 /LM) had effects similar to adenosine in that it hyperpolarized the membrane potential with a decrease in input resistance (n = 4) (Fig. 4B). When cyclic AMP reduced the a.h.p. (Fig. 4B) this reduction could be largely accounted for by the decrease in input resistance. The same concentration of 8-Br cyclic AMP (100,M) caused a clear reduction in the a.h.p. without any decrease in membrane resistance (not shown). Since these two agents, adenosine and cyclic AMP, did not mimic the actions of 8-Br cyclic AMP, and indeed,

6 250 A D. V. MADISON AND R. A. NICOLL Adenosine J~~~ B Cyclic AMP 3 min Fig. 4. Adenosine and cyclic AMP do not mimic 8-Br cyclic AMP in CAl pyramidal cells. A, chart records from a pyramidal cell bathed in TTX (1 /M) and TEA (5 mm) showing the a.h.p. following a current evoked calcium action potential. Application of adenosine (10uM) caused a hyperpolarization of the membrane potential and did not decrease the a.h.p. The membrane potential of this cell was -67 mv. B, records from another pyramidal cell recorded in the same conditions as A. The control (left record) response consisted of a hyperpolarizing current pulse followed by a depolarizing stimulus and an a.h.p. During application of cyclic AMP, hyperpolarizing current pulses alone were applied to the cell. Application of cyclic AMP (100,uM) caused a hyperpolarization of the membrane potential, accompanied by a decrease in membrane input resistance. Note that the magnitude of this conductance increase is similar in magnitude to the decrease in the amplitude of the a.h.p. (middle record). Direct current was applied through the recording electrode to return the membrane potential to base-line levels at the first arrow, and was turned off at the second arrow. The membrane potential of this cell was -63 mv. had opposite effects on resting membrane potential and input resistance, it seems unlikely that the analogue exerts its effects through an adenosine receptor. Support for an intracellular site of cyclic AMP action was provided by experiments in which cyclic AMP was injected into pyramidal neurones through the recording electrode. Such intracellular injection of cyclic AMP produced responses indistinguishable from those produced by extracellular application of 8-Br cyclic AMP or NA. While the onset of the action of intracellularly injected cyclic AMP occurred too quickly to allow the recording of control responses, all cells so injected (n = 11) showed little accommodation and fired for the entire duration of long depolarizing current pulses, and slow a.h.p.s were not recorded (Fig. 5 top). This can be compared with uninjected cells, of which more than 98 % had pronounced slow a.h.p.s (cf. Madison & Nicoll, 1984). In contrast, cells injected with the identical concentration (100 mm) of 5' AMP showed normal responses in that they accommodated to long current pulses and slow a.h.p.s were produced in response to short duration depolarizing stimuli (n = 5, Fig. 5 middle). The fact that cyclic AMP applied outside the cell had no noradrenergic fl-receptor-like effects, while intracellular injection of this same substance mimicked noradrenergic action suggests strongly that these fl-actions require that cyclic AMP act at an intracellular site.

7 CYCLIC AMP AND NORADRENALINE IN HIPPOCAMPUS ms 600 ms 10.5 na Cyclic AMP 1 5mV 04_~~~~~~~~4 mv J5 mv 1n 5' AMP j 1 ~~~~~~~~~~~~~2 na G uanylyl-i imidodiphosphate I _v Fig. 5. Intracellular injection of cyclic AMP and 5'-guanylyl-imidodiphosphate reduce calcium-activated potassium a.h.p.s and accommodation in CA1 pyramidal cells. Each row of responses in this Figure were taken from a different pyramidal cell, all bathed in normal medium. Compounds were allowed to leak from the electrode without any driving current. Top, effects of intracellular injection of cyclic AMP (100 mm) on the a.h.p. (60 ms stimulus) and on accommodation (600 ms stimulus). Membrane potential = -58 mv. Middle, effects of intracellular injection of 5'AMP (100 mm). Membrane potential = -61 mv. Bottom, effects of intracellular injection of guanylyl-imidodiphosphate (100 mm). The membrane potential of this cell was -54 mv. Direct activation of adenylate cyclase If noradrenergic f-receptor actions are mediated by cyclic AMP, then activation of the pyramidal cell's endogenous adenylate cyclase should mimic the actions of NA. Pyramidal neurones were injected with the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate which promotes adenylate cyclase activity by binding to the 'G' regulatory protein of this enzyme (Rodbell, Lin, Salomon, Londos, Harwood, Martin, Rendell & Berman, 1975). All (n = 5) neurones recorded with this substance in the electrode showed severely reduced accommodation when stimulated with a long duration current pulse, and lacked a slow a.h.p. when stimulated with a short duration pulse (Fig. 5 bottom). The diterpene compound, forskolin, was applied extracellularly to sixteen pyramidal neurones. This compound, which acts directly on the adenylate cyclase to cause its activation (Seamon & Daly, 1983), has the advantage that it can be applied extracellularly, thus allowing for control responses to be recorded. When applied to

8 252 D. V. MADISON AND R. A. NICOLL A Control Forskolin Wash i111 lililil Ill 2~~~~~~~~~~i20 mv JL _ B Fig. 6. Forskolin reduces calcium-activated potassium a.h.p.s and accommodation in CAI pyramidal cells. A, photographic record of the response of a pyramidal cell to a long duration depolarizing current pulse, taken in control, 23 min after addition of 50 /SMforskolin to the superfusing medium, and 72 min after the flow of normal medium was restored. B, chart records of the response of the pyramidal cell to a short duration depolarizing current pulse, showing the a.h.p. Records in B were taken within 15 s of those in A. Note that forskolin application reduces the slow component of the a.h.p. and simultaneously reduced accommodation of action potential discharge. The membrane potential of this cell was -66 mv. pyramidal neurones by bath application, forskolin caused a marked decrease in accommodation in the action potential train evoked by a long depolarizing current pulse (Fig. 6A). This was accompanied by a decrease in the amplitude of the slow a.h.p. elicited by a short duration stimulus (Fig. 6B). 10 /zm-forskolin caused an average reduction in the amplitude of the a.h.p. of 58% (+ 37 S.D., n = 9). Forskolin also usually caused a small depolarization of the membrane potential (not shown). Both the reduction in accommodation and the a.h.p. were reversed by washing forskolin from the bathing medium. Effects of inhibition ofphosphodiesterase and adenylate cyclase on noradrenergic actions The effect of NA on calcium-activated potassium a.h.p.s was examined under conditions of reduced phosphodiesterase activity. If the actions of NA are mediated through cyclic AMP, then reducing the activity of this enzyme should potentiate the inhibitory effect ofna on the a.h.p. Two agents were used to inhibit phosphodiesterase activity, IBMX and Ro , both of which have been shown to increase NAstimulated cyclic AMP levels in brain slice tissues (Schultz & Daly, 1973; Dismukes & Daly, 1974). The actions of these drugs were apparent alone as well as in conjunction with NA application. Both agents often caused small reductions in the amplitude of the a.h.p. when applied to the preparation, although enhancement of the action of NA could be seen in the absence of any such change in the a.h.p. Both IBMX and Ro enhanced the action ofna on the peak amplitude of the a.h.p. with 50,uM-IBMX causing an average 300 % increase in NA responses over control I 1 s 5 mnv

9 CYCLIC AMP AND NORADRENALINE IN HIPPOCAMPUS 253 A Control NA Wash B IBMX IBMX+NA Wash C Ro Ro2O NA Wash D Control NA Wash JJl10mV 2 s Fig. 7. IBMX and Ro enhance the action of NA on the a.h.p. in CAI pyramidal cells. All records shown in this Figure are from the same pyramidal cell bathed in TTX (1 JiM) and TEA (5 mm). Shown are chart records of a.h.p.s which follow current-evoked calcium action potentials. A, application of NA (1 /LM for 6 min) reduced the amplitude of the a.h.p. approximately 32 %. The a.h.p. returned to control amplitude after NA was washed from the recording chamber (10 min). B, 3-isobutyl-1-methyl xanthine (IBMX) (1 mm for 15 min) reduced the a.h.p. amplitude approximately 260% (compare with A, 'wash'). Addition of 1,sM-NA (for 4 min) to the superfusate reduced the a.h.p. 720% compared with its amplitude in IBMX alone and 79 % compared with control amplitude. The a.h.p. returned to control after washing IBMX and NA from the recording chamber for 21 min. C, application of Ro (1 mm for 19 min) reduced the amplitude of the a.h.p. approximately 15% (compare with B, 'wash'). Addition of 1 JiM-NA (for 6 min) reduced the amplitude of the a.h.p. approximately 59 % compared to its amplitude in Ro alone and 65 % compared to control amplitude. The a.h.p. returned to control amplitude 37 min after washing Ro and NA from the recording chamber. D, following the wash-out of Ro , NA was reapplied to the preparation. Application of 1 /LM-NA (for 6 min) reduced the amplitude of the a.h.p. by only 24 % compared to control. The amplitude of the a.h.p. returned to control level upon washing NA from the chamber. The membrane potential of this cell was -56 mv.

10 254 D. V. MADISON AND R. A. NICOLL A 1 Control NA Wash 2 B 1 SQ22,536 SQ22,536 + NA Wash 2 C 1 Control NA Wash 2 2 s Fig. 8. SQ22,536 inhibits the action of NA on the a.h.p. in CAI pyramidal cells. All records shown in this Figure were taken from the same pyramidal cell bathed in TTX (1 gm) and TEA (5 mm). Records shown were recorded on a Nicolet digital oscilloscope and plotted on a Hewlett-Packard digital plotter. A, application of NA (1 fum for 6 min) caused a 48 % decrease in the amplitude of the a.h.p. following a current evoked calcium action potential. The a.h.p. returned to control amplitude when NA was removed from the superfusing medium. B, application of SQ22,536 (100 FM for 36 min) caused a small (7 %) increase in the size of the a.h.p. Reapplication of NA (1 FM for 6 min) in the presence of SQ22,536 caused only an 11 % decrease in the a.h.p. amplitude. C, reapplication of NA (1 FM for 4 min), 35 min after SQ22,536 had been removed from the superfusate, caused a 39% decrease in the amplitude of the a.h.p. Responses shown in the right-hand column (A2, B2 and C2) are superimposed plots of control records, and traces recorded after NA application, to illustrate the NA-blocking effects of SQ22,536 more clearly. The membrane potential of this cell was -56 mv. (n = 5), and 100 pm-ro causing an average 275 % (n = 6) increase. In Fig. 7, a pyramidal cell was exposed to 1 /m-na which caused a reversible 32 % reduction in the amplitude of the a.h.p. (Fig. 7A). Application of IBMX (1 mm) caused a 26 % reduction in the a.h.p. and also enhanced the action of NA such that when 1 /UM-NA was reapplied it now caused a 72% inhibition of the a.h.p. (Fig. 7B). Likewise, addition of Ro (1 mm) to the same neurone caused a 15% reduction in the size of the a.h.p. and NA application (1 /SM), with Ro , caused a 59% decrease in the amplitude of the a.h.p. (Fig. 7C). These effects were reversed upon washing Ro from the recording chamber (Fig. 7D).

11 CYCLIC AMP AND NORADRENALINE IN HIPPOCAMPUS Inhibition of adenylate cyclase activity should reduce the action of NA, if cyclic AMP is its second messenger. Application of SQ22,536 (9-(tetrahydro-2-furyl) adenine), an agent which reduces the activity of cyclase in mammalian platelets (Harris, Asaad, Phillips, Goldenberg & Antonaccio, 1979) and sympathetic ganglia (Brown & Dunn, 1983), reduced the action of NA on the a.h.p. SQ22,536 by itself caused an average 200 increase in the size of the a.h.p. when applied at 100 fm (n = 8). This same concentration of SQ22,536 inhibited the action ofna in six ofeight cells tested (average effect, a 360 inhibition of NA action) and in those two cells showing no final inhibition of NA actions, the onset of the a.h.p. blockade was delayed. In Fig. 8, an example of the action of SQ22,536 is shown. Application of NA (1 /M) caused a 48 % reduction in the amplitude of the a.h.p. which returned to control size upon washing NA from the bath (Fig. 8A). Application of SQ22,536 (100 /tm) caused a 70 increase in the amplitude of the a.h.p., and when NA was reapplied, only an 11 o reduction of the a.h.p. occurred (Fig. 8B). The action of NA returned to normal after the SQ22,536 was washed from the recording chamber, such that NA (1 /M) caused a 39% decrease in the amplitude of the a.h.p. (Fig. 80). 255 DISCUSSION It has been proposed that four criteria must be satisfied to establish that a particular action of a neurotransmitter is mediated by the intracellular second messenger cyclic AMP (Bloom, 1975). These criteria, as adapted for noradrenergic action, are as follows (after Bloom, 1975): (i), NA must cause a change in cyclic AMP levels in target tissues; (ii), the change in intracellular cyclic AMP levels must precede NA-evoked physiological events; (iii), exogenous cyclic AMP should mimic the action of NA; and (iv), pharmacological agents which alter the function of cyclic AMP metabolic pathways should likewise alter the actions of NA. Evidence fulfilling the requirements of the first criterion, that NA must cause a rise in cyclic AMP levels in nervous tissues has been provided by many previous studies (for review, see Bloom, 1975). Application of NA to hippocampus (Palmer et al. 1973; Dolphin et al. 1979; Segal, 1981) causes a substantial increase in tissue cyclic AMP levels. Furthermore, these NA-induced increases of cyclic AMP levels, are caused by f-receptor activation, and in particular by activation of fl,-receptors (Dolphin et al. 1979). It has been reported that this increase in NA-stimulated cyclic AMP levels occurs primarily in neuronal cells (Segal et al. 1981). Thus, strong evidence exists that NA raises hippocampal cyclic AMP levels. The second criterion, that the rise in cyclic AMP levels must precede the physiological action of the transmitter, is more problematic because of the technical difficulties inherent in determining tissue cyclic AMP levels within the temporal limits required. However, Segal et al. (1981) have reported that the rise in cyclic AMP levels that follows NA application in hippocampal slices can occur as quickly as 20 s after application of the neurotransmitter. This is fast enough to precede the onset of the noradrenergic actions, reported in the accompanying paper, which have a slow onset, often taking minutes to develop fully, even after very brief applications of noradrenergic agonists (eg. Fig. 1A1). This study has provided evidence toward satisfying the remaining two criteria. As

12 256 D. V. MADISON AND R. A. NICOLL described in the accompanying report (Madison & Nicoll, 1986), NA has two fl-receptor-mediated actions in the hippocampus. These actions are a small depolarization of the resting membrane potential, which is associated with an increase in membrane input resistance, and a blockade of action potential evoked calciumactivated potassium a.h.p.s. To satisfy criterion (iii), application of exogenous cyclic AMP must mimic these actions of noradrenaline. Cyclic AMP, applied in the form of the membrane permeant analogue, 8-Br cyclic AMP, does in fact mimic exactly the,f-receptor-mediated actions of NA. This agent causes a small depolarization of the resting membrane potential accompanied by an increase in input resistance, and, like NA, also reduces the a.h.p. The mechanism of the blockade of the a.h.p. appears to be identical to that of NA, as 8-Br cyclic AMP does not reduce calcium-action potentials in these cells. In fact, under conditions of maximal stimulation, such as when high concentrations of cyclic AMP were injected into the cells, the base of the calcium action potential is often prolonged slightly as also happens with f-receptor stimulation. Further, during NA-induced reduction in a.h.p.s, in normal medium, there is a corresponding decrease in action potential frequency accommodation. This blockade of accommodation is also mimicked by application of 8-Br cyclic AMP and by cyclic AMP injection. All of the actions of exogeneously applied 8-Br cyclic AMP described here seem to occur by way of an action of the analogue at an intracellular site. This conclusion arises from the observations that 8-Br cyclic AMP bypasses the fl-receptor to exert its action since it is effective in the presence of high concentrations (10 /tm) of propranolol, and that extracellular application of the native form of cyclic AMP, which permeates membranes poorly, does not reproduce the fi actions of NA or the actions of 8-Br cyclic AMP. However, intracellular injection of cyclic AMP does reproduce these actions. These effects of injection are not simply artifacts of the experimental procedure since identical injection of the inactive 5'AMP has no such effect. Furthermore, the actions caused by 8-Br cyclic AMP are not mediated through activation of an adenosine receptor, since application of adenosine does not mimic its effects, and this particular cyclic AMP analogue is known to have very little action at the adenosine receptor, at least in sympathetic neurones (Henon & McAfee, 1983). Previous studies in the hippocampus (Segal & Bloom, 1974; Segal, 1981) reported that application of cyclic AMP or dibutyryl cyclic AMP caused inhibition of spontaneous activity or hyperpolarizations of the pyramidal cell membrane potential. In our experiments as well, cyclic AMP and adenosine caused hyperpolarizations of the pyramidal cell membrane potential. It has been suggested (Dunwiddie & Hoffer, 1980), in agreement with present results, that both cyclic AMP and dibutyryl cyclic AMP can cause inhibitory effects in the hippocampus by an action at extracellular adenosine receptors. Adenosine has been reported to increase cyclic AMP levels in hippocampal tissue (Segal et al. 1981; Fredholm, Jonzon, Lindren & Lindstr6m, 1982). This increase must presumably occur in cells other than CA1 pyramidal cells since our experiments find no electrophysiological evidence for adenosine-stimulated increases in intracellular cyclic AMP in pyramidal cells. A corollary to the third criterion is that activation of the cell's endogenous adenylate cyclase should also mimic NA action. This has been shown to be the case

13 CYCLIC AMP AND NORADRENALINE IN HIPPOCAMPUS in the experiments reported here. First, intracellular injection of 5'-guanylylimidodiphosphate, a stable GTP analogue which promotes cyclase activity by activating the GTP binding protein (Rodbell et al. 1975), produces NA-like effects in that pyramidal cells injected with this agent have no slow a.h.p.s and show little accommodation of action potential discharge when stimulated with a long depolarizing current pulse. Secondly, forskolin, which acts directly on the catalytic subunit of adenylate cyclase to cause its activation (Seamon & Daly, 1983), produces effects identical to NA and 8-Br cyclic AMP application. During application of forskolin, the pyramidal cell often depolarizes slightly, the a.h.p. is blocked and action potential frequency accommodation is reduced. This effect is similar to one reported in myenteric neurones where application of forskolin mimics slow serotonergic synaptic activation. In these cells, both forskolin and serotonin increase the number of action potentials evoked by depolarizing current pulses (Nemeth, Zafirov & Wood, 1984). The final criterion for cyclic AMP mediation of the action of NA, that pharmacological manipulations altering the cyclic AMP metabolic system must likewise alter NA responses, is also addressed by our experiments. First, we have used agents which inhibit phosphodiesterase activity. When IBMX or Ro were applied to pyramidal neurones, a small but consistent decrease in the size of the a.h.p. occurred. This suggests that the a.h.p. is tonically inhibited by basal levels of adenylate cyclase activity in pyramidal cells. This cyclase activity does not arise from low level stimulation of f-receptors by 'leakage' of NA from presynaptic boutons since application of propranolol generally did not increase the size of the a.h.p. Application of these phosphodiesterase inhibitors did, however, enhance the action of NA such that a low dose of the neurotransmitter which alone only caused a small decrease in the a.h.p., in the presence of IBMX or Ro , caused a larger one. Secondly, we have inhibited adenylate cyclase activity using SQ22,536 (Harris et al. 1979), which often slightly increased the base-line amplitude of the a.h.p. This finding also suggests that sufficient levels of cyclic AMP are produced in the resting state to tonically reduce the amplitude of the a.h.p. When applied in the presence of this agent, the action of NA was greatly diminished when compared to control levels in the same cell. Taken together, the observations that inhibition of the phosphodiesterase and inhibition of adenylate cyclase respectively increase and decrease the action of NA on the a.h.p., suggest that the action of NA is mediated by this second messenger. In summary: (i), extracellular application of cyclic AMP, in the form of the membrane permeant analogue 8-Br cyclic AMP, or intracellular application of cyclic AMP, mimic all aspects of the fl-receptor-mediated actions of NA, i.e. depolarization and blockade of calcium-activated a.h.p.s, reported in the accompanying paper; (ii), activation of the endogenous adenylate cyclase with forskolin or guanylylimidodiphosphate also mimics these actions of NA; (iii), inhibition of phosphodiesterase activity with IBMX or Ro increases the action of NA on a.h.p.s and inhibition of adenylate cyclase activity with SQ22,536 decreases the action of NA on a.h.p.s. We believe that these data strongly support the conclusion that cyclic AMP is the intracellular second messenger for these fl-receptor-mediated actions. The observation that the calcium-activated potassium conductance mechanism produces accommodation of action potential discharge (Madison & Nicoll, 1984) and thus controls the initiation of sodium action potentials, which are presumed to 9 PHY

14 258 D. V. MADISON AND R. A. NICOLL originate in the initial segment, suggests that this control mechanism is electrically close to the initial segment. Although NA-containing fibres are seen in the vicinity of the somata of pyramidal cells, their density is modest (Loy, Koziell, Lindsey & Moore, 1980). The present finding, that cyclic AMP mediates the action of NA, could provide an explanation for this apparent discrepancy. First, there is presumably a large amplification of the signal in the cyclic AMP cascade, and secondly, the involvement of a diffusible intracellular second messenger means that the channels controlled by NA need not be in the immediate vicinity of the subsynaptic membrane. Thus, because of the mediation of its actions by cyclic AMP, NA can modulate the physiological responsiveness ofpyramidal neurones at sites remote from noradrenergic fibre terminations. This type of modulation may also form the basis of noradrenergic action in neurones other than pyramidal cells. We thank Drs Robert Malenka and Rodrigo Andrade for their reading of the manuscript. This work was supported by a Bank of America-Giannini Foundation Fellowship to D. V. M. and NIH grants MH and MH (RSDA) and the Klingenstein Fund to R. A. N. REFERENCES BLOOM, F. E. (1975). The role of cyclic nucleotides in central synaptic function. Reviews of Physiology, Biochemistry and Pharmacology 74, BROWN, D. A. & DUNN, P. M. (1983). Cyclic adenosine 3',5'-monophosphate and f-effects in rat isolated superior cervical ganglia. British Journal of Pharmacology 79, DISMUKEs, K. & DALY, J. W. (1974). Norepinephrine-sensitive systems generating adenosine 3',5'-monophosphate: increased responses in cerebral cortical slices from reserpine-treated rats. Molecular Pharmacology 10, DOLPHIN, A., HAMONT, M. & BOCKAERT, J. (1979). The resolution of dopamine and fl1- and fl2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex. Brain Research 179, DRUMMOND, G. I. (1983). Cyclic nucleotides in the nervous system. Advances in Cyclic Nucleotide Research 15, DUNWIDDIE, T. V. & HOFFER, B. J. (1980). Adenine nucleotides and synaptic transmission in the in vitro rat hippocampus. British Journal of Pharmacology 69, FREDHOLM, B. B., JONZON, B., LINDGREN, E. & LINDSTR6M, K. (1982). Adenosine receptors mediating cyclic AMP production in the rat hippocampus. Journal ofneurochemistry 39, HARRIS, D. N., ASAAD, M. M., PHILLIPS, M. B., GOLDENBERG, H. J. & ANToNACcIo, M. J. (1979). Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives. Journal of Cyclic Nucleotide Research 5, HENON, B. K. & McAFEE, D. A. (1983). The ionic basis of adenosine receptor actions on post-ganglionic neurones in the rat. Journal of Physiology 336, HOFFER, B. J., SIGGINS, G. R., OLIVER, A. P. & BLOOM, F. E. (1972). Cyclic adenosine monophosphate mediated adrenergic synapses to cerebellar Purkinje cells. Advances in Cyclic Nucleotide Research 1, KAKIUCHI, S. & RALL, T. W. (1968). Studies on adenosine 3',5'-phosphate in rabbit cerebral cortex. Molecular Pharmacology 4, KAKIUCHI, S., RALL, T. W. & MCILWAIN, H. (1969). The effect of electrical stimulation upon the accumulation of adenosine 3',5'-phosphate in isolated cerebral tissue. Journal of Neurochemistry 16, LANTHORN, T., STORM, J. & ANDERSEN, P. (1984). Current-to-frequency transduction in CAI hippocampal pyramidal cells: slow prepotentials dominate the primary range firing. Experimental Brain Research 53, Loy, R., KOZIELL, D. A., LINDSEY, J. D. & MOORE, R. Y. (1980). Noradrenergic innervation of the adult rat hippocampal formation. Journal of Comparative Neurology 189,

15 CYCLIC AMP AND NORADRENALINE IN HIPPOCAMPUS MADISON, D. V. & NICOLL, R. A. (1982). Noradrenaline blocks accommodation of pyramidal cell discharge in the hippocampus. Nature 299, MADISON, D. V. & NICOLL, R. A. (1984). Control of the repetitive discharge of rat CAI pyramidal neurones in vitro. Journal of Physiology 354, MADISON, D. V. & NICOLL, R. A. (1986). Actions of noradrenaline recorded intracellularly in rat hippocampal CAI pyramidal neurones, in vitro. Journal of Physiology 372, NEMETH, P. R., ZAFIROV, D. & WOOD, J. D. (1984). Forskolin mimics slow synaptic excitation in myenteric neurons. European Journal of Pharmacology 101, PALMER, G. C., SULSER, F. & ROBISON, G. A. (1973). Effects of neurohumoral and adrenergic agents on cyclic AMP levels in various areas of the rat brain in vitro. Neuropharmacology 12, RALL, T. W. & SATTIN, A. (1970). Factors influencing the accumulation of cyclic AMP in brain tissue. Advances on Biochemical Psychopharmacology 3, RALL, T. W. & SUTHERLAND, E. W. (1961). The regulatory role of adenosine 3',5'-phosphate. Cold Spring Harbor Symposia in Quantitative Biology 26, RODBELL, M., LIN, M. C., SALOMON, Y., LONDOS, C., HARWOOD, J. P., MARTIN, B. R., RENDELL, M. & BERMAN, M. (1975). Role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones: Evidence for multisite transition states. Advances in Cyclic Nucleotide Research 5, SCHULTZ, J. & DALY, J. W. (1973). Accumulation of cyclic adenosine 3',5'-monophosphate in cerebral cortical slices from rat and mouse: Stimulatory effect of a- and fl-adrenergic agents and adenosine. Journal of Neurochemistry 21, SEAMON, K. B. & DALY, J. W. (1983). Forskolin, cyclic AMP and cellular physiology. Trends in Pharmacological Sciences 4, SEGAL, M. (1981). The action of norepinephrine in the rat hippocampus: intracellular studies in the slice preparation. Brain Research 206, SEGAL, M. & BLOOM, F. E. (1974). The action of norepinephrine in the rat hippocampus. I. Iontophoretic studies. Brain Research 72, SEGAL, M., GREENBERGER, V. & HOFSTEIN, R. (1981). Cyclic AMP-generating systems in rat hippocampal slices. Brain Research 213, SIGGINS, G. R., HOFFER, B. J. & BLOOM, F. E. (1971 a). Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. ILL. Evidence for mediation of norepinephrine effects by cyclic 3',5'-adenosine monophosphate. Brain Research 25, SIGGINS, G. R., OLIVER, A. P., HOFFER, B. J. & BLOOM, F. E. (1971b). Cyclic adenosine monophosphate and norepinephrine: effects on transmembrane properties of cerebellar Purkinje cells. Science 171, SUTHERLAND, E. W., 0YE, I. & BUTCHER, R. W. (1965). The action of epinephrine and the role of the adenyl cyclase system in hormone action. Recent Progress in Hormone Research 21,