373

Journal of Physiology (1991), 435, pp. 373-393 With 11 figures Printed in Great Britain

ANALYSIS OF ADENOSINE ACTIONS ON Ca2+ CURRENTS AND SYNAPTIC TRANSMISSION IN CULTURED RAT HIPPOCAMPAL PYRAMIDAL NEURONES

BY KENNETH P. SCHOLZ AND RICHARD J. MILLER From the Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, IL 60637, USA (Received 19 April 1990) SUMMARY

1. The role of adenosine receptors in reducing calcium currents (ICa) and in triggering presynaptic inhibition was studied using whole-cell patch-clamp techniques to record 'Ca and synaptic currents from the cell bodies of cultured rat hippocampal pyramidal neurones. Recordings of intracellular Ca2l using the indicator dye Fura-2 were used to obtain further insights into the actions of adenosine agonists. 2. The adenosine analogue 2-chloroadenosine (2-CA) reduced ICa in these neurones. This action was also evident when Ba2+ was used as the charge carrier through Ca2+ channels. Adenosine also reduced the influx of Ca2+ into the cell body during a depolarizing voltage-clamp pulse as measured with Fura-2. The potency of various adenosine receptor agonists was as follows: cyclopentyladenosine > cyclohexyladenosine > R-phenylisopropyladenosine > 2-CA > S-phenylisopropyladenosine, consistent with the pharmacological profile of an Al adenosine receptor. 3. The specific Al receptor antagonist cyclopentyltheophylline (CPT) blocked the actions of 2-CA on ICa in a competitive fashion. 4. The actions of 2-CA on ICa were abolished by pre-incubation of cultured cells with pertussis toxin (PTX; 250 ng/ml). Intracellular dialysis with the GTP analogue GTP-y-S (guanosine-5'-O-(3-thiotriphosphate)) enhanced the actions of 2-CA and rendered the response irreversible. 5. Excitatory postsynaptic currents (EPSCs) were recorded from pyramidal neurones under whole-cell voltage clamp by stimulating nearby neurones with an extracellular electrode. 2-CA potently and reversibly reduced the amplitude of EPSCs. This action was shown to be due to presynaptic inhibition of neurotransmitter release. 6. The order of potency of different adenosine agonists in reducing EPSCs was as follows: cyclopentyladenosine > cyclohexyladenosine > R-phenylisopropyladenosine > 2-CA > S-phenylisopropyladenosine. CPT inhibited the action of 2-CA in a competitive fashion. 7. The effects of 2-CA on synaptic transmission were abolished by pre-treatment with 250 ng/ml PTX, indicating that a PTX-sensitive G-protein is involved in this action. NIS 8430

374

K. P. SCHOLZ AND R. J. MILLER

8. These results indicate that activation of adenosine receptors does induce a reduction in ICa in hippocampal pyramidal neurones. Furthermore, this effect and the reduction of excitatory synaptic transmission by adenosine analogues are both mediated by PTX-sensitive G-proteins and have identical pharmacological properties. INTRODUCTION

Adenosine is a potent inhibitor of synaptic transmission at many synapses including motor nerve terminals (Silinsky, 1984), sympathetic (Wakade & Wakade, 1978) and central synapses (Okada & Ozawa, 1980; Dunwiddie, 1984). Adenosine can be released by field stimulation in the hippocampus, and may serve as a regulator of synaptic transmission in many brain regions (Snyder, 1985). Various actions of adenosine might account for its ability to reduce synaptic transmission. For example, adenosine and its analogues have been shown to activate a K+ current in hippocampal and striatal neurones (Trussel & Jackson, 1985; Gerber, Greene, Haas & Stevens, 1989). In addition, some reports have concluded that adenosine reduces calcium currents (ICa) in hippocampal pyramidal neurones (Proctor & Dunwiddie, 1983). However, this is controversial (Halliwell & Scholfield, 1984; Gerber et al. 1989) and the receptor type that may be involved in this action has not been reported. At excitatory synapses onto CAI hippocampal pyramidal neurones, adenosine and its analogues have both presynaptic and postsynaptic effects. These actions include presynaptic inhibition of neurotransmitter release (Okada & Ozawa, 1980; Dunwiddie & Haas, 1985; Dunwiddie & Fredholm, 1989) and activation of an inwardly rectifying K+ current in postsynaptic neurones (Trussell & Jackson, 1985; Dunwiddie & Fredholm, 1989). The role of GTP-binding proteins in coupling adenosine receptors to effector molecules has been studied to a limited degree. For example, it has been shown that activation of an inwardly rectifying K+ current by adenosine in cultured hippocampal and striatal neurones requires a pertussis toxin (PTX)-sensitive G-protein (Trussell & Jackson, 1987). In addition, the coupling of adenosine receptors to ICa in dorsal root ganglion neurones appears to require a G-protein (Gross, Macdonald & RyanJastrow, 1989). Whether this is also true in hippocampal neurones is unknown. The role of PTX-sensitive G-proteins in mediating presynaptic inhibition produced by adenosine is also unclear. While inhibition of glutamate release from cultured cerebellar cells by adenosine was blocked by PTX (Dolphin & Prestwich, 1985), recent experiments have drawn conflicting conclusions on the ability of PTX to block the actions of adenosine on synaptic transmission in the hippocampal slice (Fredholm, Proctor, Van der Ploeg & Dunwiddie, 1989; Stratton, Cole, Pritchett, Eccles, Worley & Baraban, 1989). We have performed experiments designed to begin to address some of these issues in cultures of rat hippocampal pyramidal neurones. Our results indicate that Al adenosine receptors are coupled to inhibition of ICa in a PTX-sensitive fashion and that this action may contribute to the mechanism of presynaptic inhibition.

ADENOSINE ACTION IN HIPPOCAMPlUS

375

METHODS

('ell culture The cell culture preparation used was a modification of the methods of Banker & Cowan (1977) and Banker (1980). The procedure is described extensively in Scholz, Baitinger, Schulman & Kelly (1988). The following is a description of the culture procedure. Seven days prior to preparing neuronal cell cultures, forebrains of newborn Sprague-Dawley rats were removed. These were used to form a culture of glial cells on tissue culture plates. After 1 week in culture, these cells reached 70-85 % confluency. For preparation of pyramidal neurones, hippocampi were dissected from embryonic rats at 17 days gestation. This is a point in development where the majority of the cells surviving in culture appear to be pyramidal neurones (Banker & Cowan, 1979; also see below). The hippocampi were dissociated in 0-25% trypsin in Hank's balanced salt solution (no added Mg2" or Ca2") for 25 min at 37 'C. This was followed by washing and trituration through flame-narrowed pasteur pipettes in the presence of DNAse. The cells were counted on a haemocytometer and plated in tissue culture media supplemented with 10% horse serum on 25 mm round glass cover-slips at a density of 10000-50000 cells/ml (approximately 300 ,ul per cover-slip). The cover-slips were prepared by application of three small paraplast 'feet' on the cell side of each cover-slip. This was followed by coating with 0 05 to 0 1 % poly-L-lysine made fresh in borate buffer. The cover-slips were then washed before use. The cells were allowed to plate down on the cover-slips for 2-4 h. At this time, the cover-slips were inverted over the cultures of glial cells so that the neurones were supported directly above the glial cells. Cells were maintained without antibiotics in chemically defined media (Dulbecco's modified Eagle's media) with added glutamine and N2.1 supplements (Bottenstein & Sato, 1979) in normal concentrations of K+ (5 3 mM) found in tissue culture media. After 4 days in culture, some of the cover-slips of cells were treated with 6-12 ,tm-cytosine arabinoside to retard proliferation of nonneuronal cells. Cultures prepared at 17 days gestation showed 5-10% immunoreactivity for y-aminobutyric acid (GABA) and predominantly excitatory synaptic potentials. Since most of the interneurones (except granule cells) in the hippocampus that have been described to date are inhibitory (Schwartzkroin, Scharfman & Sloviter, 1990), these results indicate that the cultures contained predominantly pyramidal cells. At this time in development, granule cells have not yet appeared in the hippocampus (Banker & Cowan, 1979) and are considerably smaller when they do. Cells demonstrating immunoreactivity for GABA were morphologically distinct and were not used in this study. Furthermore, preliminary results indicate that these cells do not respond to Al adenosine agonists (authors' unpublished observations). Cultures prepared from 18 days gestation (just 1 day later) showed about 12-36% immunoreactivity for GABA and about 50% of evoked synaptic potentials were inhibitory (K. P. Scholz & W. K. Scholz, unpublished observations). For this reason, the experiments reported here were performed on cells cultured from rats at 17 days gestation.

Electrophysiology (Cover-slips containing pyramidal neurones (in culture 7-24 days) were removed from the presence of the astrocytes and placed in a perfusion chamber on the stage of an inverted microscope. All experiments were performed at room temperature (21-23 °C). Cells were perfused at a rate of 1-15 ml/min. Recordings were obtained using a List EPC-7 (Darmstadt, Germany) patch-clamp amplifier in the whole-cell mode (Hamill, Marty, Neher, Sakmann & Sigworth 1981). Junction potentials were compensated before seal formation. Following formation of a seal (generallv greater that 2 GQ)) the patch of membrane below the pipette was ruptured to obtain access to the cell interior. For experiments with Ica, the pipette contained (in mM): CsCl, 136; MgCl2, 1; EGTA, 10; HEPES, 10; GTP, 1 and ATP, 3-6 at pH 7-15. Extracellular solutions contained (in mM): TEACl, 140; MIgCl2. 1; CaCl2, 5; HEPES, 10; glucose, 10 at pH 7-4. For experiments with ICal cells were stimulated every 15 s. Data were digitized at 2 kHz and stored for later analysis on an AT computer with a Labmaster A/D conversion board (Scientific Solutions, Solon, OH, USA). Traces are shown with linear leak subtracted. Current amplitudes cited in the text were measured from the zero-current level. Programs were written in the 'C' programming language and utilized Indec C-Lab subroutines. For synaptic transmission experiments, whole-cell recordings were obtained from potential

376

K. P. SCHOLZ AND R. J. MILLER

postsynaptic neurones while an extracellular electrode was used to stimulate putative presynaptic cells. Cells were voltage clamped at -50 mV and data was sampled at 10 kHz. Only monosynaptic connections were used in this study. Several procedures and criteria were used to isolate monosynaptic connections. All experiments were conducted in high-Mg2" (6 mM) solutions in order to decrease the size of postsynaptic responses to maintain a linear response of the postsynaptic membrane (see Nelson, Pun & Westbrook, 1986). This solution also decreased polysynaptic components. Furthermore, current flow through NY-methyl-D-aspartate (NMDA) receptor channels is greatly reduced in solutions containing elevated Mg2+ (Forsythe, Westbrook & Mayer, 1988). This served to maintain a more linear response of the postsynaptic membrane, which was beneficial because we were interested in using the postsynaptic response as a measure of presynaptic transmitter release. Postsynaptic responses were required to follow the stimulus in a strict one-forone fashion to have amplitudes that were independent of stimulus intensity above threshold and to have constant latency (generally 2-5 ms). For these experiments, sparse cultures were used to reduce the number of cell-cell contacts. Furthermore, the cover-slips on which the cells were grown were broken into small fragments before placement in the chamber. As a result, some experiments were conducted with only two cells present in the recording chamber, allowing questions about polysynaptic components to be addressed unequivocally. Alternatively, since the cultures contain only a small number of glial cells, it is possible to estimate the number of cells that a neurone may contact by visual inspection. Neurones were selected that appeared to contact only one or two other cells. Any remaining polysynaptic components were eliminated by the selection procedures described above. In addition, since other neurones were relatively distant from the desired synaptic pair (due to the sparse culture conditions) the difference in conduction time was large so that unwanted synaptic currents could be distinguished. The extracellular solution for these experiments contained (in mM): NaCl, 140; KCl, 5; CaCl2, 3; MgCl2, 6; HEPES, 10, glucose, 10 at pH 7 4. The pipette solution used in these experiments contained (in mM): potassium acetate, 140; MgCl2, 1; EGTA, 10; HEPES, 10 at a pH of 7-15. No ATP regenerating system or GTP were added to this solution. Under these conditions, the postsynaptic membrane lost much of its responsiveness to adenosine analogues (see also Trussell & Jackson, 1985, 1987), allowing for a more faithful recording of transmitter release from the presynaptic neurone. Fura-2 recording of intracellular Ca2+ Since the neurones used for these studies have extensive dendritic arborizations, it was difficult to obtain good voltage control of the membrane. For this reason, some experiments were conducted to test for the possibility that measurements of ICa were contaminated by currents arising from poorly clamped neuronal processes. For these experiments, intracellular Ca21 was measured with the indicator dye Fura-2 (Grynkiewicz, Poenie & Tsien, 1985) simultaneously with recordings of ICa' Since it has been shown that the cell bodies of pyramidal neurones can be effectively voltage clamped (Kay & Wong, 1987), these experiments serve as a control for possible contamination of ICa by currents from distal regions of the cell. In addition, some experiments were conducted with the membrane-permeable form of Fura-2 in the absence of whole-cell dialysis to test for PTXinsensitive components that may have been 'washed out' by dialysis of the cytoplasm. The apparatus used for recording of intracellular Ca2+ consisted of a light source, photomultiplier tube and a dual channel sample-and-hold circuit. Ultraviolet light from a mercury arc lamp was passed through a rotating filter wheel containing bandpass filters centred at 340 and 380 nM (Omega Optical, Brattleboro, VT, USA). The period of rotation was adjustable from 5-200 ms. This light was fed into the epi-fluorescence port of a Nikon inverted microscope and used to excite the dye in the cells. Emitted fluorescence was passed through a bandpass filter centred at 510 nm, passed through a diaphragm centred on the cell and recorded by a photomultiplier tube. This signal was further processed by sample-and-hold circuits synchronized with the rotation of the filter wheel. The sample-and-hold operation could be adjusted to average the signal for any desired time window during the passage of the appropriate filter through the light path. The data was then recorded and stored on an AT compatible computer. Two methods of Fura-2 dye loading were used. For experiments in which whole-cell recordings were performed simultaneously with measurements of intracellular Ca2 , the pipette solution contained 500 /IM-Fura-2 and the concentration of EGTA was reduced to 10 JM. Although concentrations of Fura-2 below 100 /IM provided sufficient fluorescence for the recording instrumentation, they were not satisfactory for buffering intracellular Ca2+. However, addition of

ADENVOSINE ACTION IN HIPPOCAMPUS

377

EGTA to provide sufficient buffering of Ca2" greatly reduced the response of Fura-2 to Ca2+ influx. Therefore, a relatively high concentration of Fura-2 was used to provide sufficient Ca2' buffering to maintain the activity of Ca2+ channels. For experiments that did not involve whole-cell recording. cells were loaded with Fura-2 by incubating with the acetoxvmethvl ester form (4/tM solubilized in dimethyl sulphoxide, DMSO; final D)NISO concentration 0-1 %) in buffered saline to which had been added 5 mg/ml bovine serum albumin. Cells were loaded for 45 min and washed in buffered saline for 30 min before use. Since the Fura-2 technique gives a semi-quantitative indication of intracellular free Ca2", all experiments were performed with internal control depolarizations for comparison to experimental treatments. Values for intracellular free Ca2' are approximate and given for comparison only. Calibration of Fura-2 ratios was performed as per Thayer, Sturek & Miller (1988) and converted to approximate intracellular Ca2+ concentration as per Grynkiewicz et al. (1985). Drugs Pharmacological agents were obtained from the following sources: 2 chloroadenosine, cyclopentyladenosine, R-phenylisopropyladenosine, S-phenylisopropyladenosine, cyclohexyladenosine were obtained from Sigma (St Louis. MO, USA); cyclopentyltheophylline was from Research Biochemicals Inc. (Natick, MA, USA). Fura-2 was from Molecular Probes (Eugene, OR, USA). BRL34915 (cromakalim) was a gift from Dr Michael Rogawski. 6-Cyano-7-nitroquinoxaline2,3-dione (CNQX) was from Tocris Neuramin (Essex, UK). All of these agents were added to the solution superfusing the cells at the indicated concentrations. Petussis toxin was from List Biologicals (Campbell, CA, USA) and was added to the culture dishes at a final concentration of 250 ng/ml at least 10 h prior to experiments. Although effects of PTX could be observed at concentrations down to 50 ng/ml following incubations for 5 h, the above procedure was used routinely to avoid any possible ambiguities.

RESULTS

Inhibition of calcium currents by adenosine analogues Calcium currents (ICa) were activated in cultured pyramidal neurones in whole-cell clamp by applying depolarizing voltage pulses (200 ms) from holding potentials of -80 and -40 mV to a test potential of 0 mV. The ICa of these cells with 5 mM-Ca21 as the charge carrier were generally 1-2 nA. Currents were elicited every 15 s. In cells voltage clamped to 0 mV from a holding potential of -80 mV, application of 1 ,CM2-chloroadenosine (2-CA) to the bath caused a 24 + 1P5 % (mean + S.E.M.) decrease in the amplitude of 'Ca as measured at a time corresponding to the peak current in control traces (Fig. IA). No measurable change in leak could be detected under the conditions used for these experiments. Inhibition of ICa was also evident when currents were elicited from a holding potential of -40 mV (Fig. 1B). The response was observed in all cells tested (n = 45), and appeared to show no desensitization to multiple applications of 2-CA (Fig. 1 C). The EC50 for reduction of ICa by 2-CA was about 0-6 /IM (see Fig. 4B), similar to results from dorsal root ganglion neurones (0-8 aM; Kasai & Aosaki, 1989). Due to the extensive dendritic arborizations of these neurones, it was often difficult to obtain excellent voltage control of the membrane. Although we have only analysed cells in which the voltage control appeared to be reasonable, it is still difficult to make unequivocal conclusions regarding the effects of 2-CA on the kinetics of 'Ca' as has been done in dorsal root ganglion neurones (Gross et al. 1989; Kasai & Aosaki, 1989). Indeed, it could be argued that the observed reduction in Ica actually reflects activation of K+ currents in poorly voltage clamped regions of the

K. P. SCHOLZ AND R. J. MILLER

378

cell as has been proposed by Gerber et al. (1989). Several types of experiments were performed to address this possibility. First, the K' current that is activated by adenosine is blocked by Ba2" (Gerber et al. 1989). To test whether activation of this K' current is responsible for the

A

B

Vh = -80 mV

Vh

=

-40 mV

40 ms C

0

Time (s) 200 400 600

800

-0.5 3

-1.5 -

(c -2.0 -251u -2.5 1PM

a) cl

0.1

PLM 1 ,uM

~I.I

Fig. 1. 2-Chloroadenosine (2-CA) reduces 'Ca in cultured hippocampal pyramidal neurones. A, whole-cell voltage-clamp recording of ICa activated by a depolarizing pulse (200 ms) to 0 mV from a holding potential (Vh) of -80 mV before and during application of 1 /LM-2CA. B, the current in the same cell evoked from a holding potential of -40 mV. C, plot of the peak inward current as a function of time for the two different holding potentials, (@, VH = -80 mV; 0, Vh = -40 mV). Test potential (VJ) = 0 mV. 2-CA was added to the solution superfusing the cells at the indicated concentrations for the indicated times.

reduction in ICaw experiments were performed with 2 or 5 mM-Ba2" substituted for Ca2' as the charge carrier through Ca2" channels. Figure 2A shows current responses to voltage pulses to test potentials ranging from -70 to + 40 mV from a holding potential of -80 mV with 2 mM-Ba2+ as the charge carrier. In the presence of the Al-selective adenosine agonist cyclopentyladenosine (CPA; 0 1 SUM) the current was reduced (Fig. 2B). The current-voltage (I-V) plots of these results (Fig. 2 C) indicate that CPA inhibited the current without a significant shift in the voltage dependence (n = 7). Furthermore, the inhibition of peak barium current ('Ba) at a test potential of -20 mV was 301 + 1 6 % in 0 1 aM-CPA. This compares well with the inhibition seen with 5 mM-Ca2+ as charge carrier at a test potential of 0 mV (36 + 1 1 %; n = 13). These results suggest that K' channel activation in poorly clamped regions of the cell is not responsible for the inhibition of current observed. Finally, it is noteworthy that

ADENOSINE ACTION IN HIPPOCAMPUS

379

the maximum inhibition of 'Ba was observed at the peak of the I-V curve with no accompanying shift in the extrapolated reversal potential. This is further support for direct inhibition of IBa by CPA. It has been reported that many neurotransmitters that inhibit ICa through Gprotein-coupled mechanisms slow the rate of activation of 'Ca (cf. Bean, 1989). This effect was variable when Ca2" was used as the charge carrier in our experiments. However, with 2 mM-Ba2' as the charge carrier, 0 1 /tM-CPA shifted the time to peak of IBa at a test potential of -20 mV from 23 + 2-2 ms in control traces to 58 + 6-5 ms

(n= 5). An additional test for adenosine receptor-mediated inhibition of ICa was performed by measuring Ca2` influx during a voltage-clamp pulse. Although it is possible that voltage control in axonal and dendritic processes is poor, the voltage control in the cell soma is likely to be effective (Kay & Wong, 1987). Therefore, since Ca2+ diffuses very slowly in cytoplasm (Nasi & Tillotson, 1985; Simon & Llinas, 1985), measurements of Ca2+ elevation in the cell body during brief voltage-clamp pulses should be free from contamination arising from poor voltage control of peripheral processes. This principle was used to further test the hypothesis that adenosine analogues reduce ICa in pyramidal neurones. For these experiments, Fura-2 (500 ,tM; K+ salt) was added to the pipette solution and the concentration of EGTA in the pipette was reduced to 10 /M. Following rupture of the membrane under the seal, the cell was excited by alternating 340 and 380 nm light. Data were collected and analysed as described in Methods. Figure 2D shows that CPA reduced both ICa and Ca2+ elevation induced by a voltage-clamp pulse from -80 to 0 mV (n = 3). These results indicate that voltage-dependent influx of Ca2+ is reduced by adenosine analogues.

Pharmacological profile of adenosine receptor coupled to ICa In order to determine the subtype of adenosine receptor that is responsible for reducing ICa in pyramidal neurones, various adenosine agonists were tested for their ability to reduce ICa, At least two agonists were compared in each neurone to determine the rank order of potency at a concentration of 100 nm. In one cell, which was representative of all of the cells, each of the five analogues were tested. Representative traces from this cell are shown in Fig. 3A. The results are summarized in Fig. 3B. The order of potency was insensitive to the order in which the different analogues were applied. Figure 3C shows the results of an experiment in which intracellular Ca21 was measured during applications of 50 mM-K+ saline to depolarize the cells in the absence of whole-cell dialysis. Each application was preceded by superfusion with control saline or an adenosine agonist, as indicated. The rank order of potency in reducing elevations in Ca2+ was identical to that obtained from whole-cell recording experiments (n = 3). The order was as follows: cyclopentyladenosine (CPA) > cyclohexyladenosine (CHA) > R-phenylisopropyladenosine (RPIA) > 2-CA > S-phenylisopropyladenosine (SPIA). This pharmacological profile is consistent with that of the Al subtype of adenosine receptor (Bruns, Lu & Pugsley, 1986). An additional characteristic of Al adenosine receptors is competitive blockade by cyclopentyltheophylline (CPT; Bruns et al. 1986; Dunwiddie & Fredholm, 1989). In

K. P. SCHOLZ AND R. J. MILLER

380

cultured pyramidal neurones, CPT (100 nM) reduced the potency of 1 ,tM-2-CA in reducing ICa (Fig. 4A; n = 5). An approximate pA2 value was obtained using an antagonist concentration of 0-1 ,lM and the relation pA2 = - log[B] + log[DR -1], where [B] is the concentration of antagonist and DR is the dose ratio of agonist at B 0.1 pM-CPA

Control

A

-20 mV -L

-70 mV

mV

-_~~~~~~~~

.

v

+40 mV -10 mV

C a

0o.

....

.iX

.

E

0 r-

.

;

......4

0)-1 pM-CPA 1

F380 F380

-1*0'

X -1pjM-CPA

C

a) U-

1.5

., , .,; .

D

Control

Wash

C J

-0.7 nA

0.25 nA

-80 -60 -40 -20 0 20 40 Test potential (mV)

40 ms

Fig. 2. Current responses to a series of depolarizing pulses from a holding potential of -80 mV to the indicated test potentials and the corresponding voltage traces for a cell under control conditions (A) and in the presence of 0'1 ,ZM-cyclopentyladenosine (CPA; B). The charge carrier was 2 mM-Ba2 C, plot of current at 10 ms after initiation of the test pulse from the traces shown in A and B. D, simultaneous recording of Ic. and intracellular Ca2+ in another pyramidal neurone with 5 mM-Ca2+ as the charge carrier. Addition of 0 1 /LM-CPA reduced both ICa and Ca2+ influx associated with the depolarizing pulse. Vh -80 mV, Vt 0 mV. F340/F380, ratio of fluorescence signals at 340 and 380 nm. .

=

=

the estimated EC50. The approximate pA2 for CPT action at the receptor was 8'0, similar to that reported for binding of CPT to Al receptors (Bruns et at. 1986). Although 2-CA is capable of activating both Al and A2 receptors, CPT is a highly selective antagonist at the Al receptor. Note that CPT was able to block virtually

AI)ENOSINE A(CTITON IN HJIIPOCA4PL S

381

all actions of 2-CA. inidicating that activation of A2 receptors did not participate in the inhibitorv actions of 2-CA on (Ca* Furthermore. possible non-specific actions of 2-CA (e.g. direct block of Ca21 channels) can be ruled out. These results provide further evidence that activation of the Al receptor is responsible for inhibition of 'Ca bv adleniosine receptor agonists. A

B 2 CPA o CHA

RPIA c 2-CA 0 o SPIA; 0 10 20 30 40 50 Percentage reduction (peak) c 2000 All 100 nM 1500 SPIA RPIA 2- A jI CHA

C E

+

1000

CN

500 0.1

,uM-SPIA

K=

0.5 nA 40

ms

~~~RPIA

CPAI

0 0

10

20 30 40 Time (min)

50

FiY. :3. Pharmnacological profile of the a(lenosiine receptor litnked to inhibition of I A recor(lings from a single nieuronie of the actions of 01 pmn of the following (see text for explaniation of al)breviations): 2-CA. (T'A (a = 13). RPTIA (i? = 8). CHA (n = 5). and SPIA (ai = 4). 1V =-80 mV. 1E = 0 mV. B. bar graph summarizing the results presented in . C. Ftura-2 reeordinig of 'Ca2- elevation triggered hv applications of 50 mm-K' in the ab)sence or presence of 0)1 mI various a(lenosine analogues in a cell loa(le(l with dye by inl)cation wN-ithi the membrane-permeable formll. N-ote that the responise to conitrol (lepolarizations gra(lually declines (luring the experimenit.

Role of (GI-binditp9 proteinsTI ta ativ tion of a p)otassiulmnc(ur t in (cllt ured hippocalmp)al neurones b)y adelosinew agolists (' (ad) has been sth0ownN- to be albolished by p)e-treatment with J'TX (Trussell & .Jackson. 1987 ; Zgombick. Bcck. Alahle, (raddock-Boval & Alaavanis. 1989). 1 n1(adition. the reduc(tion of tlhe' N-type I(a in dorsal I:oot gaInglionI

K. P. SCHOLZ AND R. J. MILLER

382

B

A

c3 30

1 MM-CPT 1

pM-2-CA

+4'a (D

C

20

0.1

-

ltM-CPT

10

lmMGPT

0.5 nA 40 ms

-6

-5

log[2-CA] (M)

Fig. 4. Blockade of the action of 2-CA by the At-selective antagonist cyclopentyltheophylline (CPT). A, 1 #uM-CPT nearly abolished the effect of 1 #uM-2-CA. Vh = -80 mV, V, = 0 mV. B, dose-response plot of the effects of 2-CA on ICa in the presence of different concentrations of CPT. Each point represents the mean + S.E.M of four or more determinations. A

B

GTP-y-S

-0-5-

0-1 pm-2-CA = l

Time (s) 200 400 600 800 ....... ~~~~~~~~~~~~~~0.0

0

c

2

;=0S ~~~~1nA

1.-10

-2 01

L2

pm-2-CA

tMZC

40 ms Fig. 5. Actions of GTP-y-S on ICa* A, ICa recorded in the presence of 500 ,uM-GTP-y-S in the pipette before and after addition of 0 1 ,tM-2-CA to the bath. V'h = -80 mV, V, = 0 mV. B, time course of peak 'Ca from A. *, F, = -=80 mV; 0, VJ =-40 mV. Note that the second application of 2-CA was without effect.

neurones requires a PTX-sensitive G-protein (Gross et al. 1989). In order to determine if the effects of Al adenosine receptor activation on Ica in cultured pyramidal neurones was also mediated through a G-protein, the active nonhydrolysable analogue of GTP, GTP-y-S(guanosine-5'-O-(3-thiotriphosphate)) was included in the patch pipette. Inclusion of 500 ,tM-GTP-y-S in the continued presence of 1 mM-GTP in the patch pipette increased the magnitude of the effects of subsaturating concentrations of 2CA to 46+ 15% reduction (Fig. 5A; n = 5). In addition, GTP-y-S rendered the effects of 2-CA irreversible and occluded the effects of subsequent applications of agonist (Fig. 5B). These results indicate that a G-protein is essential in coupling Al adenosine receptors to inhibition of Ica in cultured hippocampal pyramidal neurones. Certain G-proteins are substrates for ADP ribosylation by a process catalysed by PTX. This leads to inactivation of the G-protein (Ui, Katada, Murayama, Kurose, Yajima, Tamura, Nakamura & Nogimori, 1984). Therefore we tested the sensitivity

ADENOSINE ACTION IN HIPPOCAMPUS

383

of the effects of 2-CA to PTX. Cultures were pre-treated with PTX (250 ng/ml) 16-42 h prior to experimental analysis. Figure 6 A demonstrates that pre-treatment with PTX abolished the response to 2-CA in voltage-clamped cells (n = 5; no measurable inhibition observed in any cells). These results indicate that the G-protein involved in coupling Al receptors to inhibition of ICa is most likely of the Gi or G. subclass, since these are sensitive to PTX (Ui et al. 1984). To test the possibility that a PTX-insensitive component of the response exists that is washed out by intracellular dialysis, cells were loaded with the membranepermeable Fura-2 acetoxymethyl ester. Pulses of 50 mM-K' were used to depolarize cells in the absence of intracellular dialysis, as described above. The cells demonstrated robust increases in intracellular free Ca2" (Fig. 6B). Following application of 1 jtM-2-CA, the response to an identical application of 50 mM-K+ was attenuated by 21 + 3-2 % (n = 17 of 17 cells tested). In addition, the ability of 2-CA to reduce Ca2+ influx stimulated with 50 mM-K+ was blocked by pre-treatment with PTX (Fig. 6 C; n = 8; on average, a small increase of 1 + 0-9 % was observed). These results suggest that no PTX-insensitive component is present that may have been washed out by dialysis during whole-cell recording experiments.

Characterization of synaptic transmission and presynaptic inhibition induced by 2-CA The actions of adenosine on synaptic transmission were studied by recording from postsynaptic neurones while an extracellular electrode was used to stimulate presynaptic neurones (see Methods). A current-clamp recording from a presynaptic cell during stimulation with this technique demonstrated that the stimulus (50-200 ,uA; 100 Its) elicited a single action potential (Fig. 7A). As shown, some cells underwent a hyperpolarization before firing an action potential, thereby prolonging the latency between the stimulus and the firing of the presynaptic cell. Although we have demonstrated that adenosine analogues reduce ICa in the cell body, Fig. 7A demonstrates that addition of 1 cM-2-CA had no detectable effects on the shape of the action potential in the cell body of the presynaptic neurone (see Discussion). A small hyperpolarization (1-2 mV) was observed in some cells. Measurements of K+ currents activated by adenosine revealed that 2-CA activated a small current (24-40 pA; data not shown), presumably the same current described previously (Trussell & Jackson, 1985). This current is considerably smaller than its counterpart in the hippocampal slice preparation (Okada & Ozawa, 1980; Gerber et al. 1989; see Discussion). Recordings of excitatory postsynaptic currents were conducted in solutions containing 3 mm-Ca2+ and 6 mM-Mg2+ in order to reduce polysynaptic components and to reduce the amplitude and non-linearity of postsynaptic responses. Only monosynaptic excitatory connections were used for these experiments as determined by constant-latency, one-for-one excitatory postsynaptic currents (EPSCs) recorded in the postsynaptic neurones (see Methods). Addition of 0-1 ,tM-2-CA to the bath reduced the amplitude of the postsynaptic current in a consistent and reversible fashion (Fig. 7B and C; n = 18). These experiments are complicated by possible postsynaptic effects of the neurotransmitter, thereby making it difficult to draw conclusions about presynaptic

K. P. SCHOLZ AND R. J. MILLER

384

inhibition. We have developed an empirical procedure to assess the relative contributions of pre- and postsynaptic actions to the observed effects of a neurotransmitter. The stimulus intensity applied to the extracellular electrode, as well as the location of the extracellular electrode were adjusted in each experiment A

400r

B

Control PTX 16 h 300 CN

1

pM-2-CA

100

0

1

pM-2-CA

40 ms 0

C

300

5

15 10 Time (min)

20

PTX 13 h

200 CN

cu

100 1 pM-2-CA

0

0

5

10 15 Time (min)

20

Fig. 6. Pertussis toxin (PTX) blocks the ability of 2-CA to inhibit ICa. A, ICa recorded before and during application of 1 1tM-2-CA in a cell pre-treated with 250 ng/ml PTX for 16 h. Vh = -80 mV, V, = 0 mV. B, recording of intracellular Ca2+ in the absence of wholecell recording using Fura-2 axetoxymethyl ester. Example of a control cell demonstrating inhibition of Ca2+ influx by 2-CA. C, recording of a cell pre-treated with PTX for 13 h. The effects of 2-CA were blocked under these conditions.

to yield both an antidromic stimulus (arising from antidromic spread of dendritic depolarization into the voltage-clamped soma due to direct stimulation of the peripheral processes of the postsynaptic cell) and an orthodromic stimulus (reflecting activation of synaptic transmission following stimulation of an action potential in the presynaptic neurone). The amplitude of the antidromic stimulus was a graded function of the stimulus intensity whereas the synaptic current appeared in an allor-none fashion. Both of these responses were recorded in the postsynaptic cell; the synaptic current follows the antidromic stimulus by a constant latency when the synaptic connection is monosynaptic. Under the recording conditions used for these experiments, the EPSCs were completely and reversibly blocked by addition of 6-

ADENOSINE ACTION IN HIPPOCAMPUS

385

cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10/tM; Fig. 8A), indicating that they were due to activation of quisqualate and/or kainate subtypes of glutamate receptors (Honore, Davies, Drejer, Fletcher, Jacobsen, Lodge & Nielsen, 1988). The antidromic stimulus was used to assess any changes that a neurotransmitter or other substance may have induced in the membrane properties of the postsynaptic C B

A

c

CI 2 ms 5ins

mV a 0.25 nA

5D u

0 0.0-

Time (s) 100 200

300

-0.1-0.3-

0.p1uM-2-CA. B, whole-cell vmp2rcA Cs r0o synaptictransmission following2addition of 01CAmM-2-CA to t0e ba D

al

extracellular stimulation with a bipolar electrode before and during (arrow) application of,lam-2-CA. B, whole-cell voltage-clamp recording of EPSCs resulting from extracellular stimulation of a presynaptic neurone. These traces shows the progressive inhibition of mV. -a50 synaptic transmission following addition of0an sym-2-CA to the bath. =ac C, time course of peak synaptic current from B. D, the adenosine Al agonist CPA had no measurable effect on IPSCs.

cell, as well as random fluctuations that may occur. Figure 8B demonstrates that addition of cromakalim (BRL 34915), which has been shown to increase K+ currents in hippocampal neurones (Alzheimer, Sutor, ten Bruggencate, 1989; Politi, Suzuki & Rogawski, 1989), reduced both the antidromic and synaptic currents and increased the latency to the EPSC. This suggests that cromakalim has a major postsynaptic component to its action (i.e. increased conductance of the postsynaptic neurone leading to shunting of both antidromic and synaptic currents and a decrease in conduction velocity). In contrast, 2-CA (1 tm) reduced the synaptic current by 75% with no detectable effect on the antidromic current (Fig. 80), indicating that the effect of 2-CA was primarily presynaptic. Both antidromic and orthodromic responses were monitored in each experiment and cells demonstrating any postsynaptic effects were discarded. The recordings shown in Fig. 7A of a presynaptic neurone were obtained with patch electrodes that contained ATP and GTP. Under these conditions IK,.n can be observed during the entire recording period. in contrast, the remainder of the recordings from postsynaptic neurones were obtained with no ATP or GTP in the pipette. Under these conditions IK adn disappeared within 5-10 min after commencement of whole-cell recording (see Trussel & Jackson, 1985); however, the 13

PHY 435

K. P. SCHOLZ AND R. J. MILLER

386

synaptic inhibition we have observed is insensitive to the absence or presence of GTP in the postsynaptic cell. This provides additional evidence that the synaptic inhibition observed is not due to activation of IK adn in postsynaptic cells.

When cultures were prepared from fetal rats a day later than usual (i.e. 18 days gestation) the number of inhibitory synaptic potentials greatly increased (see A 10 Mm-CQNX Wash

Control

B2

B1

10

,uM-cromakalim

-__| 10 gM-cromakalim

C2

C1

1 pM-2-CA

f 2 nA

__

1 !LM-2-CA

0.25 nA

5 ms

Fig. 8. Properties of synaptic transmission. A, addition of 10 ,tM-CNQX abolished EPSCs in a readily reversible manner. B 1, in another cell, addition of 10 ,LM-cromakalim reduced the antidromic response of the postsynaptic cell (see double arrow). B2, higher gain recording of trace in B 1 showing that cromakalim also reduced the amplitude of EPSCs and increased the latency (see single arrow), indicating a postsynaptic locus of action. C 1, in another cell, addition of 1 ,uM-2-CA had no effect on the antidromic stimulus (double arrow). C2, higher gain recording of trace in Cl showing that this concentration of 2-CA progressively reduced the EPSC without affecting latency (single arrow), indicating a presynaptic locus of action. V7h = -50 mV throughout.

ADENOSINE ACTION IN HIPPOCAMPUS 387 Methods). The presynaptic character of the inhibition of synaptic transmission was further supported by the observation that, in these cultures prepared from more developed fetuses, CPA had no effect on inhibitory postsynaptic currents (JPSCs) recorded in the same neurones in which EPSCs were reduced by 95 % (Fig. 7D). Since a

2-CA I

0.5 nA

5 ms

c

b

RPIA

SPIA

__1 e

d

CHA

CPA

Fig. 9. Pharmacological profile of presynaptic inhibition induced by adenosine analogues. EPSCs recorded in the absence or presence of 0 1 #uM-2-CA (n = 18), SPIA (n = 6), RPIA (n = 7), CHA (n = 3), or CPA (n = 9). a,b and c are from one cell d and e from another. Vh = -50 mV throughout.

a postsynaptic effect leading to activation Of IK adn would be expected to reduce both EPSCs and IPSCs in a similar fashion, this finding indicates that a presynaptic mechanism underlies the reduction of EPSCs induced by adenosine analogues.

Synaptic inhibition produced by adenosine analogues In order to compare the adenosine receptor-induced reduction of ICa to that inducing presynaptic inhibition, the relative potencies of various adenosine analogues in reducing EPSCs were determined using an agonist concentration of 0.1 /LM. Representative examples for each agonist are shown in Fig. 9. The relative potencies of these compounds were as follows: CPA > CHA > RPIA > 2-CA > SPIA. This pharmacological profile is identical to that found for reduction Of ICa and is consistent with the profile of the Al receptor. This suggests that the same receptor may mediate these two actions. In some cells, the postsynaptic currents appeared to have several components all satisfying the 13-2

K. P. SCHOLZ AND R. J. MILLER

388

criteria for a monosynaptic connection. While in some cases this may be due to multiple monosynaptic connections from different cells, it was apparent that in many cases this was due to multiple synaptic sites from a single cell. Indeed, the recordings shown in Fig. 9 were obtained from pairs of cells that were on small fragments of cover-slips in the chamber. In these examples, these A

0.1 pM-CPT C

+

1 pM-2-CA

0-1 pM-CPT

100 co

(2)

_

/ \ -60 |~ ~ ~~~~~: 1.

B

(6) pm--CPLT (18

~,40OL80-

5ins 1 pm-2-CA

CD0

C

-8

-6

-7

log

-5

-4

[2-CAl (M)

Fig. 10. Blockade of the actions of 2-CA by CPT. A, EPSCs recorded in the presence of 0-1 1tM-CPT before and during application of 1#UM-2-CA. B, in the same cell after washing out CPT, addition of I #M-2-CA had a much greater effect. VJ = -50 mV. C, dose-response plots of the actions of 2-CA in the absence or presence of 01 or 0 5 4aM-CPT. Each point represents the mean + S.E.M. of three determinations unless otherwise indicated in parentheses. were the only cells present in the experimental chamber. Since other axons can be seen to degenerate within minutes of being severed from the cell body, it is likely that the multiple components seen in these traces originate from a single presynaptic cell that synapses onto different locations of the postsynaptic neurone.

Additional characterization of the adenosine receptor linked to presynaptic inhibition was performed by using the Al-selective antagonist cyclopentyltheophylline CPT. In some cells, CPT caused a small increase in the amplitude of EPSCs (not shown), possibly indicating a tonic inhibition mediated by endogenous adenosine. The actions of 2-CA were blocked by CPT (Fig. lOA; n = 10) with an approximate pA2 of 80-841 (Fig. lOB). This value is close to that obtained for inhibition of ICa (8*0), lending further support to the possibility that these two actions are mediated by the same adenosine receptor. Two recent reports have assessed the sensitivity to pertussis toxin (PTX) of presynaptic inhibition induced by adenosine in the hippocampus (Fredholm et al. 1989; Stratton et al. 1989). These reports have come to conflicting conclusions on this matter. To address this issue further, we have tested the PTX sensitivity of presynaptic inhibition produced by adenosine. Pre-incubation of cultures with 250 ng/ml of PTX for 13-48 h completely abolished the actions of 2-CA on synaptic transmission (Fig. 11; n = 7). Of further interest is the observation that no additional effects were uncovered in the presence of PTX, as has been reported in a

ADENOSINE ACTION IN HIPPOCAMPUS

389

neuromuscular preparation (Silinsky, Solsona & Hirsh, 1989), although the concentrations of agonist that were used in these experiments may lead to only weak activation of non-Al receptors. These results are consistent with those of Stratton et al. (1989), who reported that injection of PTX directly into the hippocampus abolished the effects of 2-CA on synaptic transmission PTX 13 h

1

um-2-CA_

nA ~~~~~0.5

|

L

5 ms

Fig. 11. Pre-incubation of cells with PTX abolished the ability of 2-CA to induce presynaptic inhibition. Vh =-50 mV. DISCUSSION

Adenosine receptor activation reduces ICa Previous reports have demonstrated that activation of adenosine receptors can reduce the duration of Ca2' action potentials in hippocampal pyramidal neurones (Proctor & Dunwiddie, 1983) However, the mechanisms underlying this effect are unclear. Some reports have concluded that adenosine does not reduce Ca2+ spikes or currents in these cells (Halliwell & Scholfield, 1884; Gerber et al. 1989). Indeed, it has been suggested previously that the reduction in Ca2+ spikes is due solely to activation of a K+ current by adenosine in poorly space-clamped neuronal processes (Gerber et al. 1989). We have extended these observations by measuring ICa and Ca2+ influx using whole-cell voltage clamp and simultaneous recording of fluorescence from the Ca2+ indicator dye Fura-2. Given that Ca2+ diffuses very slowly in cytoplasm (Nasi & Tillotson, 1985; Simon & Llinas, 1985), measurements of intracellular Ca2+ in the soma of a voltage-clamped neurone should be virtually free from contamination due to poorly space-clamped neuronal processes. This is especially true for measurements of intracellular Ca2+ during brief depolarizing pulses, since the Ca2+ will not diffuse appreciably during the pulse. The results of Fig. 2 demonstrate that the influx of Ca2+

390

K. P. SCHOLZ AND R. J. MILLER

during a 200 ms depolarizing pulse to 0 mV is reduced in the presence of the Alselective agonist CPA. Therefore, we conclude that activation of adenosine receptors does indeed reduce ICa in hippocampal pyramidal neurones. Many neurotransmitters that act through PTX-sensitive G-proteins reduce the Ntype ICa in neurones (for review see Tsien, Lipscombe, Madison, Bley & Fox, 1988). For example, Gross et al. (1989) showed that the reduction of ICa by 2-CA in dorsal root ganglion neurones is specific for the N-type current. Since the cultured pyramidal neurones used in this study are difficult to voltage clamp, it is difficult to make such a conclusion with this preparation. However, the results presented are consistent with the previous findings mentioned above (see also Madison, Fox & Tsien, 1987). In addition, adenosine analogues often slowed the rate of activation of IBa (cf. Fig. 2), although this is variable when Ca2+ is used-as the charge carrier. This may be a result of the phenomenon described by Bean (1989) and by Kasai & Aosaki (1989) in which the voltage dependence of activation of ICa is altered by neurotransmitters. Pharmacology of adenosine receptor There are at least three defined binding sites for adenosine in neuronal tissue. Two of these are external receptors, the Al and A2 receptors, and the third is an intracellular binding site called the 'P' site. In addition, it is possible that nucleotides may inhibit ICa non-specifically. All of the evidence presented here indicates that reduction of ICa and presynaptic inhibition are mediated by the Al adenosine receptor. The effects are present at nanomolar concentrations, show the expected order of potency for Al agonists, demonstrate stereoselectivity between the 'S' and 'R' stereoisomers of phenylisopropyladenosine (PIA), and are blocked by the Al-selective antagonist CPT at concentrations near those reported for binding to Al receptors. We conclude that the receptor linked to inhibition of ICa is indistinguishable from that linked to presynaptic inhibition on the basis of currently available ligands. Experiments conducted on mouse dorsal root ganglion neurones in culture demonstrated that the adenosine receptor that inhibited ICa in these cells showed little stereospecificity between the S and R isomers of PIA (Macdonald, Skerritt & Werz, 1986). This would not be predicted from binding studies on central neurones. The question was raised whether this might also be true for reduction of ICa in hippocampus, or perhaps in many tissues. The pharmacological profile of the receptor linked to inhibition of ICa in cultured hippocampal pyramidal neurones was consistent with that reported in binding studies (Fig. 3). We obtained no evidence of an additional receptor type on these cells. We do not know whether the different results arise from differences between dorsal root ganglion (DRG) and hippocampal neurones or arise from species differences between mouse and rat.

Cellular mechanism of adenosine action The role of G-proteins in mediating the actions of adenosine receptors has been somewhat controversial. While several reports have concluded that IK, adn is blocked by PTX (Trussell & Jackson, 1987; Fredholm et al. 1989; Stratton et al. 1989; Zgombick et al. 1989), there have been conflicting reports regarding the sensitivity of

ADENOSINE ACTION IN HIPPOCAMPUS

391

presynaptic inhibition to PTX, especially in the hippocampal slice preparation. One recent report has concluded that this confusion may arise from the differing effectiveness of PTX injected into different locations in the brain. When injected into the third ventricle, PTX appears to block the actions of adenosine on IKadn in postsynaptic neurones while having no effect on presynaptic inhibition (Fredholm et al. 1989). However, injection directly into the hippocampus blocks both actions (Stratton et al. 1989). It seems likely that the toxin has difficulty reaching the presynaptic sites responsible for mediating presynaptic inhibition in this case. The cell culture preparation is useful in this regard, as there is little to hinder the access of PTX to different parts of the cell. Inhibition of glutamate release from cultured cerebellar neurones by adenosine is blocked by pre-treatment with PTX (Dolphin & Prestwich, 1985). However, it is difficult to determine the role of presynaptic inhibition, as distinct from reductions in ICa or excitability, in such a case. The role of G-proteins in the responses to adenosine receptor activation in cultured pyramidal neurones is demonstrated in Figs 5, 6 and 11. Pre-incubation of cells with pertussis toxin, which inactivates Go and Gi subtypes of G-proteins, blocked the actions of adenosine analogues on ICa and on synaptic transmission. These results are consistent with those of Gross et al. (1989) who reported the PTX sensitivity of 2-CA actions on ICa in DRG neurones, and of Stratton et al. (1989), who injected PTX directly into the hippocampus and found blockade of presynaptic inhibition of EPSCs in CAI neurones. We would like to thank Dr W. K. Scholz for preparing neuronal cultures and Drs A. P. Fox and M. McCarren for helpful comments on an earlier version of the manuscript. This work was supported by PHS training grant T32-NS07195-07, PHS grants DA02121, DA02575 and MH40165 and by grants from Marion and Miles Laboratories. W. K. S. was supported by GM42715. REFERENCES

ALZHEIMER, C., SUTOR, B. & TEN BRUGGENCATE, G. (1989). Effects of cromakalim (BRL 34915) on potassium conductances in CA3 neurons of the guinea-pig hippocampus in vitro. NaunynSchmiedeberg's Archives of Pharmacology 340, 465-471. BANKER, G. A. (1980). Trophic interactions between astroglial cells and hippocampal neurons in culture. Science 209, 809-810. BANKER, G. A. & COWAN, W. M. (1977). Rat hippocampal neurons in dispersed cell culture. Brain Research 126, 397-425. BANKER, G. A. & COWAN, W. M. (1979). Further observations on hippocampal neurons in dispersed cell culture. Journal of Comparative Neurology 187, 469-494. BEAN, B. P. (1989). Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340, 153-156. BOTTENSTEIN, J. E. & SATO, G. H. (1979). Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proceedings of the National Academy of Sciences of the USA 76, 514-519. BRUNS, R. F., Lu, G. H. & PUJSLEY, T. A. (1986). Characterization of the A2 adenosine receptor labeled by 3H-NECA in rat striatal membranes. Molecular Pharmacology 29, 331-346. DOLPHIN, A. C. & PRESTWICH, S. A. (1985). Pertussis toxin reverses adenosine inhibition of neuronal glutamate release. Nature 316, 148-150. DUNWIDDIE, T. V. (1984). Interactions between the effects of adenosine and calcium on synaptic responses in rat hippocampus in vitro. Journal of Physiology 350, 545-559. DUNWIDDIE, T. V. & FREDHOLM, B. B. (1989). Adenosine Al receptors inhibit adenylate cyclase activity and neurotransmitter release and hyperpolarize pyramidal neurons in rat hippocampus. Journal of Pharmacology and Experimental Therapeutics 249, 31-36.

392

K. P. SCHOLZ AND R. J. MILLER

DUNWIDDIE, T. V. & HAAS, H. L. (1985). Adenosine increases synaptic facilitation in the in vitro rat hippocampus: evidence for a presynaptic site of action. Journal of Physiology 369, 365-377. FORSYTHE, I. D., WESTBROOK, G. L. & MAYER, M. L. (1988). Modulation of excitatory synaptic transmission by glycine and zinc in cultures of mouse hippocampal neurons. Journal of Neuroscience 8, 3733-3741. FREDHOLM, B. B., PROCTOR, WV., VAN DER PLOEG, 0. & DUNWIDDIE, T. V. (1989). In vivo pertussis toxin treatment attenuates some, but not all adenosine Al effects in slices of the rat hippocampus. European Journal of Pharmacology 172, 249-262. GERBER, U., GREENE, R. W., HAAS, H. L. & STEVENS, D. R. (1989). Characterization of inhibition mediated by adenosine in the hippocampus of the rat in vitro. Journal of Physiology 417, 567-578. GROSS, R. A., MACDONALD, R. L. & RYAN-JASTROW, T. (1989). 2-Chloroadenosine reduces the N calcium current of cultured mouse sensory neurones in a pertussis toxin-sensitive manner. Journal of Physiology 411, 585-595. GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca2l indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450. HALLIWELL, J. V. & SCHOLFIELD, C. N. (1984). Somatically recorded Ca-currents in guinea-pig hippocampal and olfactory cortex neurones are resistant to adenosine action. Neuroscience Letters 50, 13-18. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. (1981). Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfiuigers Archiv 391, 85-100. HONORE, T., DAVIES, S. N., DREJER, J., FLETCHER. E. J., JACOBSEN, P., LODGE, D. & NIELSEN, F. E. (1988). Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonist. Science 241, 701-704. KASAI, H. & AOSAKI, T. (1989). Modulation of Ca-channel current by an adenosine analog mediated by a GTP-binding protein in chick sensory neurons. Pfiugers Archiv 414, 145-149. KAY, A. R. & WONG, R. K. S. (1987). Calcium current activation kinetics in isolated pyramidal neurones of the CAI region of the mature guinea-pig hippocampus. Journal of Physiology 392, 603-616. MACDONALD, R. L., SKERRITT, J. H. & WERZ, M. A. (1986). Adenosine agonists reduce voltagedependent calcium conductance of mouse sensory neurones in cell culture. Journal of Physiology 370, 75-90. MADISON, D. L., Fox, A. P. & TSIEN, R. W. (1987). Adenosine reduces an inactivating component of calcium current in hippocampal CA3 neurons. Biophysical Journal 51, 30a. NASI, E. & TILLOTSON, D. (1985). The rate of diffusion of Ca2l and Ba2+ in a nerve cell body. Biophysical Journal 47, 735-738. NELSON, P. G., PUN, R. Y. K. & WESTBROOK, G. L. (1986). Synaptic excitation in cultures of mouse spinal cord neurones: receptor pharmacology and behaviour of synaptic currents. Journal of Physiology 372, 169-190. OKADA, Y. & OZAWA, S. (1980). Inhibitory action of adenosine on synaptic transmission in the hippocampus of the guinea pig in vitro. European Journal of Pharmacology 68, 483-492. POLITI, D. M. T, SUZUKI, S. & ROGAWSKI, M. A. (1989). BRL 34915 (cromakalim) enhances voltage-dependent K' current in cultured rat hippocampal neurons. European Journal of Pharmacology 168, 7-14. PROCTOR, W. R. & DUNWIDDIE, T. V. (1983). Adenosine inhibits calcium spikes in hippocampal pyramidal neurons in vitro. Neuroscience Letters 35, 197-201. SCHOLZ, W. K., BAITINGER, C., SCHULMAN, H. & KELLY, P. T. (1988). Developmental changes in Ca2" calmodulin-dependent protein kinase II in cultures of hippocampal pyramidal neurons and astrocytes. Journal of Neuroscience 8, 1039-1051. SCHWARTZKROIN, P. A., SCHARFMAN, H. E. & SLOVITER, R. S. (1990). The Hippocampal Region as a Model for the Study of Brain Structure and Function, ed. STORM-MATHISON, J, ZIMMER, J. & OTTERSEN, 0. P., pp. 269-286. Elsevier, Amsterdam. SILINSKY, E. M. (1984). On the mechanism by which adenosine receptor activation inhibits the release of acetylcholine from motor nerve endings. Journal of Physiology 346, 243-256. SILINSKY, E. M., SOLSONA, C. & HIRSH, J. K. (1989). Pertussis toxin prevents the inhibitory effect of adenosine and unmasks adenosine-induced excitation of mammalian motor nerve terminal. British Journal of Pharmacology 97, 16-18.

ADENVOSINVE ACTION IN HIPPOCAMPUS

393

SIMON, S. M. & LLINA~S, R. R. (1985). Compartmentalization of the submembrane calcium activity during calcium influx and its significance to transmitter release. Biophysical Journal 48, 485-498. SNYDER, S. H. (1985). Adenosine as a neuromodulator. Annual Review of Neuroscience 8, 103-124. STRATTON, K. R., COLE, A. J., PRITCHETT, J., ECCLES, C. U., WORLEY, P. F & BARABAN, J. M. (1989). Intrahippocampal injection of pertussis toxin blocks adenosine suppression of synaptic responses. Brain Research 494, 359-364. THAYER, S. A., STUREK, M. & MILLER, R. J. (1988). Measurement of neuronal Ca2" transients using simultaneous microfluorimetry and electrophysiology. Pftuigers Archiv 412, 216-222. TRUSSELL, L. 0. & JACKSON, M. B. (1985). Adenosine-activated potassium conductance in cultured striatal neurons. Proceedings of the National Academy of Sciences of the USA 82, 4857-4861. TRUSSELL, L. 0. & JACKSON, M. B. (1987). Dependence of an adenosine-activated potassium current on a GTP-binding protein in mammalian central neurons. Journal of Neuroscience 7, 3306-3316. TSIEN, R. W., LIPSCOMBE, D., MADISON, D. V., BLEY, K. R. & Fox, A. P. (1988). Multiple types of calcium channels and their selective modulation. Trends in Neurosciences 11, 431-438. UI, M., KATADA, T., MURAYAMA, T., KUROSE, T., YAJIMA, M., TAMURA, M., NAKAMURA, T. & NoGIMORI, K. (1984). Islet activating protein pertussis toxin: a specific uncoupler of receptormediated inhibition of adenylate cyclase. Advances in Cyclic Nucleotide Research 17, 145-151. WAKADE, A. R. & WAKADE, T. D. (1978). Inhibition of noradrenaline release by adenosine. Journal of Physiology 282, 35-49. ZGOMBICK, J. M., BECK, S. G., MAHLE, C. D., CRADDOCK-ROYAL, B. & MAAYANI, S. (1989). Pertussis toxin-sensitive guanine nucleotide-binding proteins(s) couple adenosine Al and 5hydroxytryptamine lA receptors to the same effector systems in rat hippocampus: Biochemical and electrophysiological studies. Molecular Pharmacology 35, 484-494.