- Email: [email protected]
Effects of bivalent cations on adenosine sensitivity in the rat hippocampal slice
Brain Research, 617 (1993) 61-68
61
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 19014
Effects of bivalent cations on adenosine sensitivity in the rat hippocampal slice D.A. Smith
a
and T.V. D u n w i d d i e
b,c
a Neuroscience Program, Oberlin College, Oberlin, OH 44074 (USA), b Department of Pharmacology and Program in Neuroscience, University of Colorado Health Sciences Center, Denver, CO 80262 (USA) and c Veterans Administration Medical Center, Denver, CO 80220 (USA)
(Accepted 15 February 1993)
Key words: Adenosine; A1 receptor; Hippocampus; Ca2+; Mg2+; Presynaptic inhibition; Synaptic modulation
Ligand binding studies have demonstrated that bivalent cations can alter interactions between purinergic agonists and adenosine receptors in brain membranes. In the present study, we have investigated whether a similar interaction can be demonstrated in terms of a functional response to adenosine, which is the inhibition of synaptic transmission in the CA1 region of the rat hippocampus mediated via presynaptic A1 receptors. Our data suggest that alterations in cation concentrations do not significantly affect the sensitivity of these adenosine receptors, as long as changes are made in such a way as to leave presynaptic Ca 2+ entry unaffected. The experimental results do not support the conclusion that there is a specific effect of either Ca z+ or Mg2+ on adenosine receptor sensitivity, such as has been described for agonist interactions with adenosine A1 receptors. We conclude that the effects of bivalent cations observed in ligand binding studies probably reflect an effect at an intracellular site, either on the receptor itself, or perhaps on associated GTP binding proteins.
INTRODUCTION A d e n o s i n e is an important n e u r o m o d u l a t o r 10,26 that both inhibits transmitter release at a variety of central and peripheral synapses 7"14'15A7, and has direct depressant actions on postsynaptic n e u r o n s 19'3°. Both the preand postsynaptic actions of adenosine have b e e n observed in the CA1 region of the hippocampus, a region where adenosine blocks the release of glutamate and hyperpolarizes pyramidal cells 3'4'2°'24'25. T h e s e effects of adenosine are consistent with evidence demonstrating that adenosine A1 receptors are located presynaptically on the axon terminals of CA3 pyramidal cells, and postsynaptically on the dendrites of CA1 pyramidal cells 5. T h e inhibition of transmitter release may involve inhibition of presynaptic Ca 2+ influx 14'22'23, while the postsynaptic effects are primarily related to the activation of a G-protein c o u p l e d potassium channel 29'3°. It is also significant that while adenosine inhibits synaptic transmission at virtually all excitatory synapses in the h i p p o c a m p a l formation, it does not inhibit transmission at G A B A e r g i c synapses is. Thus, the net effect of adenosine in this brain region is to
depress excitatory transmission and reduce postsynaptic excitability, while leaving inhibitory circuits largely unaffected. Because of the p r o f o u n d inhibitory effect that adenosine exerts over electrophysiological activity in the CA1 region, and because there appears to be a continuous basal level of adenosine receptor activation s , it has b e e n suggested that adenosine may be an important tonic regulator of neural activity in the hipp o e a m p u s l°. Conversely, the excitatory effects of methylxanthines such as caffeine and theophylline have b e e n attributed to their ability to block adenosine receptors, and thereby disrupt this tonic inhibitory effect of adenosine 1°'~2. Factors that might regulate this basal activity, such as changes in adenosine receptor number, or changes in the sensitivity of adenosine receptors, could have important effects on electrophysiological activity. In this context, previous reports that bivalent cations such as Ca 2+, Mg 2+, and Mn 2+ can e n h a n c e the specific binding of adenosine receptor agonists (but not antagonists) to adenosine receptors 16'21'31 suggests that this could be a significant m e c h a n i s m by which adenosine sensitivity is regulated.
Correspondence: D. Smith, Neuroscience Program, Sperry Hall, Oberlin College, Oberlin, OH 44074, USA.
62 S o m e p r e v i o u s e l e c t r o p h y s i o l o g i c a l s t u d i e s in t h e hip-
s p i k e is e x t r e m e l y s e n s i t i v e to a g e n t s t h a t c h a n g e c e l l u -
p o c a m p u s h a v e r e a c h e d a s i m i l a r c o n c l u s i o n as well, in
lar excitability, a n d r e s p o n d s in a n o n - l i n e a r f a s h i o n to
t h a t an a p p a r e n t r e d u c t i o n in t h e sensitivity to a d e n o -
c h a n g e s in s y n a p t i c input. F o r t h e s e r e a s o n s , t h e p o p u -
sine is o b s e r v e d
l a t i o n s p i k e is n o t an a c c u r a t e
under
conditions where
the
M g 2+
c o n c e n t r a t i o n in t h e r e c o r d i n g m e d i u m is r e d u c e d or e l i m i n a t e d J,2,27,2~. T h e r e are at least t h r e e p o s s i b l e m e c h a n i s m s t h a t
strength.
indicator of synaptic
T h i s is p a r t i c u l a r l y t r u e in t h e c a s e o f an
a g e n t such as a d e n o s i n e , w h i c h w o u l d b e e x p e c t e d to r e d u c e t h e p o p u l a t i o n spike by v i r t u e o f its ability to
m i g h t e x p l a i n this result. First, as p r o p o s e d by S t o n e
h y p e r p o l a r i z e p y r a m i d a l n e u r o n s , i n d e p e n d e n t l y o f any
a n d c o l l e a g u e s ~, t h e r e m a y be a s p e c i f i c e f f e c t o f M g 2+
e f f e c t s it m i g h t h a v e on s y n a p t i c t r a n s m i s s i o n .
( a n d possibly C o ~+) ions o n a d e n o s i n e
receptors
to
f a c i l i t a t e a d e n o s i n e r e s p o n s e s . A s e c o n d possibility t h a t
MATERIALS AND METHODS
is m o r e in a c c o r d a n c e w i t h t h e l i g a n d b i n d i n g s t u d i e s o f a d e n o s i n e r e c e p t o r s ~6'213~ is t h a t t h e r e is a g e n e r a l ized e f f e c t o f b i v a l e n t c a t i o n s , such t h a t C a " , M g " , o r M n 2+ a n d o t h e r s i m i l a r c a t i o n s can all e n h a n c e a d e n o s i n e action. Finally, as we h a v e p r e v i o u s l y sugg e s t e d 9'~ J, t h e sensitivity o f t r a n s m i t t e r r e l e a s e to m o d u l a t i o n by a d e n o s i n e m i g h t d e p e n d
u p o n the a m o u n t
o f C a 2+ e n t e r i n g t h e n e r v e t e r m i n a l , such t h a t at high C a 2+ c o n c e n t r a t i o n s ,
the effect of adenosine
is re-
d u c e d , w h e r e a s at low C a 2+ c o n c e n t r a t i o n s , t h e e f f e c t o f a d e n o s i n e is e n h a n c e d . In o r d e r
to
determine
which
of these
potential
m e c h a n i s m s is r e s p o n s i b l e for t h e o b s e r v e d e f f e c t s o f b i v a l e n t c a t i o n s on a d e n o s i n e sensitivity, we h a v e c o n ducted
experiments
where
we
have
systematically
c h a n g e d the c o n c e n t r a t i o n s o f C a 2+ a n d M g 2+ in t h e m e d i u m , b u t in such a way t h a t t h e t h r e e p r o p o s e d hypotheses would predict differing effects on adenosine sensitivity. Specifically, we h a v e m a n i p u l a t e d bivalent cation concentrations
in s u c h a w a y t h a t M g 2+
( a n d C a 2+) c o n c e n t r a t i o n s c o u l d b e c h a n g e d w i t h o u t simultaneously
affecting
lease. F u r t h e r m o r e , encountered
basal
neurotransmitter
re-
to a v o i d s o m e o f t h e d i f f i c u l t i e s
in p r e v i o u s s t u d i e s o f b i v a l e n t c a t i o n /
a d e n o s i n e i n t e r a c t i o n s ~'2'27'2s, w e h a v e u s e d t h e f E P S P r e c o r d e d in t h e s t r a t u m r a d i a t u m o f t h e C A 1 r e g i o n as o u r r e s p o n s e m e a s u r e , r a t h e r t h a n t h e p o p u l a t i o n spike response.
It is well e s t a b l i s h e d
that
the population
Transverse, 400 ~m thick, hippocampal slices were obtained from 6- to 8-week-old~ male Sprague-Dawley rats (Sasco Animal Laboratories, Omaha, NE) using methods previously described t3. Briefly, after dissecting the hippocampus from the whole brain, slices were cut with a tissue chopper (Sorvall) and were subsequently transferred to an 0 2/COrartificial cerebral spinal fluid (ACSF) interface holding chamber to equilibrate. The chambers were maintained at 33°C. At least 1 h after surgery, the slices were transferred to a submersion recording chamber ( 1 ml volume) where they were placed on a nylon net and superfused (2 ml/min) with ACSF containing (in raM): NaC1 124. KCI 4.0, KH2PO a 1.0, MgCI.6H20 2.4, CaCIz.2H20 2.5, ~)-glucose 10, and NaHCO 3 26, pH 7.4. The perfusion medium was gassed with humidified 95% 0 2 / 5 % CO 2 and maintained at a temperature of 33-34°C. Extracellular recordings were made using glass microelectrodes (2-4 MO) filled with 2 M NaCI. fEPSPs were recorded by placing the microelectrodes in the stratum radiatum of the CA1 region of the hippocampus. Orthodromic fEPSPs were evoked every 3/1 s using a twisted bipolar nichrome wire electrode which was placed in the stratum radiatum near the border of the CAI and CA2 regions. Stimulation consisted of 0.2 ms square wave pulses. Stimulation voltage was adjusted individually for each slice to produce a fEPSP that was approximately 80% of the maximum response attainable at the start of the experiment. All electrodes were positioned visually. Only slices in which the fEPSP exceeded 1 mV were used for study. Responses were recorded using an AC amplifier, and a computer was used to digitize and store the responses for further analysis. Adenosine hemisulfate (Sigma) dissolved in distilled H 2 0 as a 5 mM stock solution was added to the recording medium via a calibrated syringe pump (Sage). The dose-response relationship for adenosine was determined by exposing slices to incrementally increasing doses (20, 50, 811 and 110 tzM) of adenosine. Each dose was applied in the recording medium for a minimum of 10 min. To assess the effects of reduced Mg 2+ levels on the adenosine dose-response curve, ACSF with the appropriate Mg 2+ and Ca 2+ concentrations was perfused in the recording chamber for at least 111-15 min before assessing the effects of adenosine. The 10-15 min
TABLE 1 The calculated and experimentally determined fEPSP amplitudes for the 4 different types of recording medium used in these experiments
All percentages are related to the amplitude of the response in solution 1 (2.5 mM Ca2+/2.4 mM Mg z+ ). The estimated fEPSP amplitude for each solution was determined as a percent of control using the formula described in the Materials and Methods section. The measured fEPSP amplitudes represent the 80% of maximal response (control ACSF), or for solutions 2-4, the amplitude of the response was set to 80% of maximum in the control ACSF, and the slice was then superfused with one of the alternate buffers and the response amplitude determined once the response had stabilized in the new medium. None of the differences between control and solutions 2-4 were statistically significant. Medium
No. ~)f slices
Ca 2 + conc. (raM)
I (Control) 2 3 4
24 14 16 9
2.5 2.0 1.5 1.5
Mg 2 + conc. (mM)
2.4 1.32 0.25 0
Estimated fEPSP amplitude
JEPSP amplitude (mV)
fEPSP amplitude (percent of control medium)
100% 10(1% 10/)% 115.2%
1.84_+(I.20 1.88 +_(I.28 1.83 + 11.28 2.37+0.31
100 _+10.9% 102.2 _+15.2% 99.5 + 15.2% 128.8_+ 16.8%
63 waiting period was chosen because preliminary observations showed that it took 5-10 min of perfusion with each of the ACSF media before stable changes in fEPSP amplitudes and waveforms were obtained. Typically, for each slice, a dose-response curve was determined in both the standard ACSF (solution 1 below) as well as in at least one of the other types of medium. The order of exposure to different media was varied in a pseudo-random fashion. In total, 4 different ACSF solutions, each with varying concentrations of Mg 2÷ and Ca 2+ were used: (1) 2.5 mM Ca 2+ and 2.4 mM Mg 2÷ (standard ACSF), (2) 2.0 mM Ca 2÷ and 1.32 mM Mg 2+, (3) 1.5 mM Ca 2+ and.25 mM Mg 2+, and (4) nominally Mg2+-free solution containing 1.5 mM Ca 2÷. The concentrations of the other constituents of the ACSF were unaltered from the values used in the standard perfusion medium. The concentrations of Ca 2÷ and Mg 2÷ chosen for all except the Mg2+-free medium were concentrations that should be equieffective in terms of transmitter release, using the relation proposed by Dodge and Rahamimoff 6 for the effects of Ca 2+ and Mg 2+ on EPSP amplitude at the frog neuromuscular junction:
EPSP = K
W [Ca] [Ca] [Mg] 1+ +-K1 K2
where K and W are constants, and K 1 and K 2 are dissociation constants for Ca 2+ and Mg 2+, respectively. As a first approximation, we used the same values for Kj and K 2 determined empirically for the neuromuscular junction (1.1 and 2.97). Preliminary studies indicated that the relationship shown above holds equally well for the hippocampal synapses under study (see Table I).
RESULTS In the present study, we sought to determine whether the sensitivity of field excitatory postsynaptic responses (fEPSPs) to inhibition by adenosine was affected by the concentrations of bivalent cations in the medium. Because it has been demonstrated that the adenosine sensitivity of fEPSP responses is affected by the basal level of neurotransmitter release 9, it was necessary to conduct these studies under conditions where transmitter release was unaffected by the changes in Ca 2+ and Mg 2+ concentrations. The Ca 2+ and Mg 2+ concentrations in solutions 1-3 were estimated to be equieffective in releasing transmitter based upon data from the frog neuromuscular junction, but we wished to demonstrate directly that this was the case in our hippocampal slice experiments. Table I summarizes data comparing the amplitudes of the fEPSPs evoked in the 4 ACSF solutions used in this study. The amplitudes of the fEPSPs evoked in solutions 1-3, which were estimated to have equal Ca 2+ activity, were virtually identical, although the fEPSPs in solutions with lower cation concentration were more likely to show evoked afterdischarges. We were unable to record responses reliably in an equivalent medium that was completely Mg2+-free, because of the extensive spontaneous and evoked bursting that occurred in this medium. Therefore, we used a MgZ+-free buffer with a higher than estimated Ca 2+ concentration (solution 4); as expected,
~
A
~ "~
Ca =* 2.5 Mg =" 2.4
V
ca ~" 1.s M g 2" 0
B Ca 2° 2.5 Mg 2" 2.4
I
1
~
a2" 1.5 Mg2" 0 1 mV 2 msee
Fig. 1. Effects of changing bivalent cation concentrations on evoked responses. The slice illustrated in A was superfused sequentially with 4 different buffers containing the indicated concentrations of Ca 2+ and Mg 2+. As can be seen, there were minor changes in the initial aspects of the response (presynaptic fiber spike and falling phase of the fEPSP), but the longer latency aspects of the waveforms showed marked changes related to firing of the pyramidal neurons. These changes are particularly pronounced in the Mg2+-free medium (bottom record). B: shows recordings from a slice that was switched from the control medium directly to the Mg2*-free buffer; again, while there was relatively little effect on the early part of the response, the later components were markedly affected. Bar = 2 ms and 1 inV.
the fEPSP amplitudes evoked in this buffer were higher than in control slices (Table I). However, under these conditions, the fEPSPs were greatly distorted by repetitive afterdischarges that altered both the shape and the latency of the recorded response (Fig. 1). In such slices, it was impossible to evoke fEPSPs of more than a few hundred microvolts amplitude without eliciting large inverted population spike responses in the dendritic recording. Such inverted population spikes can be observed in slices under control conditions, but only at very high stimulation intensities, and more than one such response is usually not observed. These high amplitude repetitive spikes suggest that the pyramidal neurons are highly excitable in this medium, probably as a result of the loss of the charge-shielding effects of Ca 2+ and Mg 2+ ions bound to the membrane. Because of this distortion, it was difficult to analyze these results in the same manner as the other responses. Clearly, the responses recorded in low or Mg2+-free media are complex and their shapes are distorted, making peak amplitude measures quite unreliable. Accordingly, in the experiments in which the sensitivity to adenosine was assessed, we also measured the initial slope of the fEPSP, an aspect of the fEPSP unlikely to
64 be contaminated by afterdischarges (see below). It was also apparent in many (Fig. 1A) but not all (Fig. 1B) slices, that the amplitude of the presynaptic fiber spike increased in amplitude as Mg 2+ and Ca 2+ concentrations were reduced. However, we did not observe any systematic differences in adenosine sensitivity between slices in which the fiber spike was increased in low Mg 2+ and Ca 2+ conditions, and those slices in which this effect was not observed. Fig. 2 presents representative examples of fEPSPs recorded in medium containing 2.5 mM Ca 2+/2.4 mM Mg 2+, and in the Mg2+-free ACSF, and illustrates the effects of adenosine on these responses. The examples recorded in standard ACSF illustrate the fact that both the amplitude of the fEPSP and the initial slope of this response were inhibited in a concentration-specific manner by adenosine superfusion. In the slices in
Mg2+-free medium, adenosine also inhibited the response; this was readily apparent in terms of the initial slope of this potential, but changes in the maximum amplitude of the fEPSP response could not be determined reliably because of the effects of inverted population spike responses. In some of the slices maintained in Mg2+-free medium, it appeared that low concentrations of adenosine actually increased the inverted population spike responses, while higher concentrations invariably abolished this component of the response. The effects of adenosine on fEPSP amplitude in the 3 ACSF solutions with approximately equivalent effective calcium concentrations are summarized in Fig. 3. In each case, adenosine reduced the peak amplitude of the fEPSP significantly (F2,.~ ~ = 241, P < 0.001) and in a concentration-dependent manner. However, the depressant effects of adenosine on fEPSP amplitude did
C
A
110 uM 110 uM 80 uM 50 uM 20 uM
80 uM 50 uM 20 uM
Control Control
D
B
110 uM 110 uM 80 uM
80 uM
50 uM
50 uM
20 uM Control
20 uM Control
Fig. 2. Effects of adenosine on field potentials in control medium, and in medium containing 1.5 m M Ca 2+ and 0 mM Mg 2+. The lower 5 records in each panel correspond to averaged responses recorded during superfusion with 110, 80, 50, 20, and 0 txM adenosine, whereas the upper record shows the same 5 averages superimposed. A and B are from slices maintained in control medium; in responses that were not contaminated by inverted population spikes (as in A), the EPSP could be measured by determining the peak amplitude of the response (indicated by vertical line), whereas in slices in which an inverted spike was observed (seen as a positive-going potential superimposed upon the negative fEPSP in B), the response was measured on the initial aspect of the fEPSP. T h e vertical line superimposed on the records in B through D indicates the time point at which m e a s u r e m e n t s of fEPSP slope were made. C and D illustrate responses evoked in Mg2+-free medium, in which multiple positive-going peaks were superimposed upon the fEPSP response. The records in B and D were obtained from the same slice but in different media. The ICs~~ values determined for these slices for adenosine were 38 txM, 41 p~M, 77 ~ M , and 36 txM for the slices shown in A - D , respectively.
65 lOO
~.
,m
/zM, and 44 + 3.7/zM in buffer solutions 1-4, respectively.
so
CI2.5/Mg 2.4 Ca2.01Mg1.32
eo
~g
,o
o uu
20
o
20 uM
50 uM 80 uM ADENOSINE CONCENTRATION
110 uM
Fig. 3. Effect of adenosine on peak fEPSP amplitude. The adenosine-mediated depression of fEPSP amplitude (mean+S.E.M.) is shown for media 1-3, which were approximately equivalent in supporting neurotransmitter release. The fEPSP amplitudes were measured as the peak negativity whenever possible under each condition. Every concentration of adenosine tested had essentially the same effects on the fEPSP response in each of the buffers.
not differ between the 3 media ( F 6 , 1 4 3 = 0.479, P < 0.822). Because of the difficulties in measuring the fEPSP in Mg2+-free buffer, we reanalyzed all of the experiments by measuring the slope of the early initial phase of the EPSP response, before any distortion induced by firing of the CA1 pyramidal neurons. Conc e n t r a t i o n - r e s p o n s e curves for adenosine under each of these conditions are shown in Fig. 4. Statistical analysis of these data reveals no concentration × ACSF condition interaction (F3,14 4 = 0.913, P < 0.515). The IC50 values were 42 + 3.2 /xM, 40 + 3.6 /xM, 36 + 2.7
1 O0 IJJ (2.
q
.-o--
Ca2.5/Mg2.4
•- e - -
Ca2.0/Mgl.3
8o
w z "3 w
60
,n
40
z uJ 0 w a.
20
DISCUSSION
Ca1.51Mg.25
T
.-O-.- C a 1 . 5 / M g . 2 5 .5/MgO
20 ADENOSINE
50 CONCENTRATION
100 (uM)
Fig. 4. Dose-response curves for adenosine. Log dose-response curves are shown for adenosine-mediated depression of the fEPSP in each of the buffers tested; the measurement in each case was the slope of the fEPSP, not the maximum amplitude. Each point represents the mean _+S.E.M. for 24 (2.5 mM Ca2+/2.4 mM Mg2+), 14 (2.0 mM Ca2+/1.32 mM Mg2+, 16 (1.5 mM Ca2+/.25 mM Mg2+), and 9 (1.5 mM Ca2+/0.0 mM Mg2+ slices tested with the indicated concentration of adenosine. IC50 values for the 4 conditions were 42 /zM, 40/~M, 36/xM, and 44 #M, respectively. There was no overall effect of buffer composition on the IC50 values determined in this manner. A few of the individual points in the display were offset slightly to enhance clarity.
Biochemical studies have demonstrated that bivalent cations can modify the interactions between agonist ligands and G-protein-coupled transmitter receptors. However, there are few functional studies that would support the hypothesis that cations are physiologically relevant modifiers of receptor sensitivity. We have examined whether the modulation of neurotransmitter release mediated via hippocampal adenosine A1 receptors shows this type of cation sensitivity. However, these experiments are complicated by the fact that the response that is measured (transmitter release), is itself a Ca2+-dependent process, and will be changed by alterations in bivalent cation concentrations. Therefore, we have developed and tested three not necessarily exclusive hypotheses that could account for bivalent cation effects on adenosine sensitivity. The first hypothesis is that there is a Mg2÷-specific effect on adenosine receptors, such that both ligand binding and functional responses are enhanced by Mg 2+. A second hypothesis, which is perhaps more in accord with the data from radioligand binding studies 16'21'31, is that a variety of bivalent cations, including Ca e ÷, MgZ ÷, Mn z +, Co 2÷, Sr 2÷, and Ba e+, can all enhance adenosine binding and functional responses, albeit with somewhat different potencies and efficacies. A third alternative, which we have proposed based upon our previous electrophysiological studies, is that the sensitivity of neurotransmitter release to inhibition by adenosine is dependent upon the amount of Ca 2+ entering the nerve terminal; at low levels of Ca 2÷ entry, adenosine is more potent in inhibiting release, whereas its potency drops with higher levels of Ca 2÷ influx. Differentiating between these alternative hypotheses is somewhat difficult, because every bivalent cation that has been demonstrated to modulate agonist binding to adenosine receptors also competes with Ca 2÷ for the presynaptic Ca 2÷ channels that mediate neurotransmitter release. For this reason, it has been necessary to develop protocols whereby the effects of cations on neurotransmitter release per se can be distinguished from possible effects on adenosine receptors. The results of the present investigations would clearly seem to rule out the first hypothesis, viz., that there is a Mg2+-specific effect on adenosine A1 receptor-mediated responses. In these experiments, we have altered the Mg 2÷ concentration in the medium by a factor of almost 10, with no detectable effect on the potency of adenosine in inhibiting synaptic transmis-
66 sion. Although there are technical difficulties involved in recording from slices in nominally Mg2+-free medium, it appeared that even under these conditions, adenosine sensitivity was not markedly altered. Whether the fEPSP slope or amplitude was used as the measure of adenosine's effect, synaptic responses were essentially abolished with the same concentration of adenosine regardless of the Mg 2+ concentration, supporting the conclusion that the effects of adenosine are not markedly attenuated by reduced Mg 2+. The second hypothesis (that the total concentration of bivalent cations regulates adenosine sensitivity) would also seem to be ruled out by these experiments as well, because changing from medium containing a total of 4.9 mM of bivalent cations to 1.75 mM had no significant effect upon sensitivity. Furthermore, in a previous study 9 we have demonstrated that increasing the Ca 2+ concentration of the medium from 1.0 to 10 mM (with Mg 2+ fixed at 2.4 raM) actually decreases adenosine sensitivity, the opposite of what would be predicted by the second hypothesis. Thus, the present findings would not seem to support previous studies suggesting that bivalent cations modify agonist interactions with adenosine A1 receptors. However, it should be stressed that there is little evidence for a bivalent cation effect on adenosine receptors in intact systems; the evidence upon which these hypotheses are based comes primarily from studies of adenylyl cyclase and ligand binding, which are conducted in membrane preparations, not in intact cells. Furthermore, there is considerable evidence to suggest that Mg 2+ effects may occur as a result of interactions with GTP-binding proteins 3~, which are thought to be intracellular, not extracellular proteins. For these reasons, we conclude that the bivalent cation effects that have been described in binding and cyclase studies probably reflect an interaction with an intracellular site, and that the present studies have not revealed effects upon adenosine sensitivity because changing the cation concentrations in the extracellular buffer has little effect upon the intracellular concentrations. As far as the third hypothesis is concerned, i.e. that adenosine sensitivity is inversely related to the amount of Ca 2+ entering the nerve terminal, the present results are consistent with predictions based upon this hypothesis. This hypothesis would suggest that if the bivalent cation concentrations are manipulated so that there is no net effect of changes in Mg 2+ and Ca 2+ concentrations on transmitter release, then no changes would be expected in adenosine sensitivity, and this was in fact the result that was observed. In the one situation where transmitter release was not equivalent (in Mg2+-free medium), the synaptic response was
somewhat greater, and the modulatory effect of adenosine correspondingly reduced, although this effect was small. This type of action was demonstrated more clearly in a previous study, where manipulations of Ca :+ and Mg 2+ that resulted in changes in the basal release of transmitter-produced highly significant changes in the adenosine sensitivity of the response 9. The basis for the Ca 2+ sensitivity of the adenosine response is not clear, but might reflect the intrinsic Ca ~+ dependency of the release process. At low levels of extracellular Ca z+, the release of transmitter varies as a 4th order function of the Ca z+ concentration, which means that very small changes in Ca 2+ have highly significant effects upon release, whereas at higher concentrations, the release process appears to saturate, such that increases in Ca z+ have less and less additional effect. If adenosine modulates the activity of a presynaptic Ca 2+ channel, or the Ca 2+ sensitivity of some aspect of the release process, then its effects should be much greater at lower rather than higher levels of Ca z+. An alternative explanation based upon the consequences of non-linear summation of EPSPs at these synapses has also been proposed (see ref. 9 for a discussion of this possibility), and would also be in accordance with the observations in the present study. One report whose results initially seem inconsistent with the general conclusions reached in this study is that of Bartrup and StoneJ; this study reported that reducing the Mg z+ concentration of the medium appeared to reduce the adenosine sensitivity of evoked population spikes in hippocampus. However, there are several aspects of this study that make this conclusion equivocal. First, these experiments assessed the effect of changing Mg 2+ on presynaptic adenosine receptors not by measuring the amplitude of the fEPSP response, which provides a direct measure of synaptic current, but rather by measuring the population spike produced by stimulation of the excitatory inputs to the pyramidal neurons. Because the population spike reflects postsynaptic cell firing, it is impossible to determine whether the changes produced by reducing Mg 2+ reflect increased cellular excitability resulting from changes in bivalent cation concentration, changes in postsynaptic adenosine receptor sensitivity, or changes in presynaptic adenosine sensitivity. The initial slope of the fEPSP response would be sensitive to only the last of these three actions, and is the only direct means to demonstrate changes in presynaptic adenosine receptor sensitivity unconfounded by other effects of bivalent cations. Because the population spike was used as the response measure in Bartrup and Stone's study, this also meant that it was not possible to adequately control for the changes in EPSP amplitude induced by changing the
67
Mg 2+ concentration, because the EPSP was never directly measured; normalizing the population spike response by reducing Ca 2+ does not accomplish this, because the population spike response reflects not only the strength of the synaptic input, but also changes in pyramidal neuron excitability. In addition, this study only investigated the effects of reductions in Mg 2+ concentration on adenosine sensitivity, whereas the binding data clearly indicate that Ca 2+ is, if anything, even more effective than Mg 2+ in enhancing adenosine receptor binding; if this were the case, then one would predict that reducing Ca 2+ concentrations should also reduce adenosine sensitivity, and in fact the opposite result appears to be the case 9. In summary, we conclude that most if not all of the available data suggest that the sensitivity of presynaptic adenosine receptors to adenosine in the CA1 region of the hippocampus is unaffected by changes in Mg 2+ concentrations. However, any manipulation that alters the Ca 2+ influx into the presynaptic nerve terminal (and correspondingly alters basal transmitter release as well), alters the apparent sensitivity of the release process to inhibition by adenosine. This effect is observed not only at high levels of Ca z+ entry, where the release mechanism might be 'saturated' with Ca 2+, but at lower levels of release as well. The functional significance of the effects of bivalent cations on adenosine receptor binding are currently unclear. If, as we hypothesize, this reflects the actions of cations at an intracellular site, and possibly on G-proteins rather than the receptor itself, then this might provide an intracellular site at which the sensitivity of the adenosine system could be regulated. Further studies will be required to demonstrate whether or not this is the case. Acknowledgements. This research was supported by Grant NS29173 (T.V.D.) and the VA Medical Research Service.
REFERENCES 1 Bartrup, J.T. and Stone, T.W., Interactions of adenosine and magnesium on rat hippocampal slices, Brain Res., 463 (1988) 374-379. 2 Bartrup, J.T. and Stone, T.W., Presynaptic actions of adenosine are magnesium-dependent, Neuropharmacology, 27 (1988) 761763. 3 Burke, S.P. and Nadler, J.V., Regulation of glutamate and aspartate release from slices of the hippocampal CA1 area: effects of adenosine and baclofen, J. Neurochem., 51 (1988) 1541-1551. 4 Corradetti, R., Lo Conte, G., Moroni, F., Passani, M.B. and Pepeu, G., Adenosine decreases aspartate and glutamate release from rat hippocampal slices, Eur. J. Pharmacol., 104 (1984) 19-26. 5 Deckert, J. and Jorgensen, M.B., Evidence for pre- and postsynaptic localization of adenosine A1 receptors in the CA1 region of the rat hippocampus: a quantitative autoradiographic study, Brain Res., 446 (1988) 161-164.
6 Dodge, F.A. and Rahamimoff, R., Co-operative action of calcium ions in transmitter release at the neuromuscular junction, J. Physiol., 193 (1967) 419-432. 7 Dolphin, A.C. and Archer, E.R., An adenosine agonist inhibits and a cyclic AMP analogue enhances the release of glutamate but not GABA from slices of rat dentate gyrus, Nature, 43 (1983) 49-54. 8 Dunwiddie, T.V., Endogenously released adenosine regulates excitability in the in vitro hippocampus, Epilepsia, 21 (1980) 541-548. 9 Dunwiddie, T.V., Interactions between the effects of adenosine and calcium on synaptic responses in rat hippocampus in vitro, J. Physiol., 350 (1984) 545-559. 10 Dunwiddie, T.V., The physiological role of adenosine in the central nervous system, Int. Rev. Neurobiol., 27 (1985) 63-139. 11 Dunwiddie, T.V. and Haas, H.L., Adenosine increases synaptic facilitation in the in vitro rat hippocampus: evidence for a presynaptic site of action, J. Physiol., 369 (1985) 365-377. 12 Dunwiddie, T.V., Hoffer, B.J. and Fredholm, B.B., Alkylxanthines elevate hippocampal excitability: evidence for a role of endogenous adenosine, Naunyn Schmiedebergs Arch. Pharmacol., 316 (1981) 326-330. 13 Dunwiddic, T.V. and Lynch, G.S., Long-term potentiation and depression of synaptic responses in the rat hippocampus: localization and frequency dependency, J. Physiol., 276 (1978) 353-367. 14 Fredholm, B.B. and Dunwiddie, T.V., How does adenosine inhibit transmitter release?, Trends Pharmacol., 9 (1988) 130-134. 15 Fredholm, B.B. and Hedqvist, P., Modulation of neurotransmission by purine nucleotides and nucleosides, Biochem. Pharmacol., 29 (1980) 1635-1643. 16 Goodman, R.R., Cooper, M.J., Gavish, M. and Snyder, S.H., Guanine nucleotide and cation regulation of the binding of [3H]cyclohexyladenosine and [3H]diethylphenylxanthine to adenosine A1 receptors in brain membranes, Mol. Pharmacol., 21 (1982) 329-335. 17 Harms, H.H., Wardeh, G. and Mulder, A.H., Adenosine modulates depolarization induced release of 3H-noradrenaline from slices of rat brain neocortex, Eur. J. Pharmacol., 49 (1978) 305308. 18 Lambert, N.A. and Teyler, T.J., Adenosine selectively depresses excitatory synaptic transmission in area CA1 of the rat hippocampus, Neurosci. Lett., 122 (1991) 50-52. 19 Lin, Y. and Phillis, J.W., Characterization of the depression of rat cerebral cortical neurons by selective adenosine agonists, Brain Res., 540 (1991) 307-310. 20 Proctor, W.R. and Dunwiddie, T.V., Adenosine inhibits calcium spikes in hippocampal pyramidal neurons in vitro, Neurosci. Lett., 35 (1983) 197-201. 21 Reddington, M., Alexander, S.P., Erfurth, A., Lee, K.S. and Kreutzberg, G.W., Biochemical and autoradiographic approaches to the characterization of adenosine receptors in brain. In E. Gerlach and B.F. Becker (Eds.), Topics and Perspectiues in Adenosine Research, Springer, Berlin, 1987, pp. 49-58. 22 Scholz, K.P. and Miller, R.J., Analysis of adenosine actions on calcium currents and synaptic transmission in cultured rat hippocampal pyramidal neurones, J. Physiol., 435 (1991) 373-393. 23 Schubert, P., Heinemann, U. and Lee, K.S., Differential effect of adenosine on pre- and postsynaptic calcium fluxes, Brain Res., 376 (1986) 382-386. 24 Segal, M., Intracellular analysis of a postsynaptic actions of adenosine in the rat hippocampus, Eur. J. Pharmacol., 79 (1982) 193-199. 25 Siggins, G.R. and Schubert, P., Adenosine depression of hippocampal neurons in vitro: an intracellular study of dose-dependent actions on synaptic and membrane potentials, Neurosci. Lett., 23 (1981) 55-60. 26 Stone, T.W., Adenosine in the Nervous System, Academic Press, San Diego, 1991. 27 Stone, T.W. and Bartrup, J.T., Magnesium-dependency of presynaptic inhibition by adenosine. In J.A. Ribeiro (Ed.), Adenosine Receptors in the Neruous System, Taylor and Francis, London, 1989, pp. 87-94.
68 28 Stone, T.W., Connick, J.H. and Bartrup, J.T., NMDA-receptorindependent effects of low magnesium: involvement of adenosine, Brain Res., 508 (1990) 333-336. 29 Trussell, L.O. and Jackson, M.B., Adenosine-activated potassium conductance in cultured striatal neurons, Proc. Natl. Acad. Sci. USA, 82 (1985) 4857-4861.
30 Trussell, L.O. and Jackson, M.B., Dependence of an adenosineactivated potassium current on a GTP-binding protein in mammalian central neurons, J. Neurosci., 7 (1987) 3306-3316. 31 Yeung, S.M., Fossom, L.H., Gill, D.L. and Cooper, D.M., Magnesium ion exerts a central role in the regulation of inhibitory adenosine receptors, Biochem. J., 229 (1985) 91-100.
61
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 19014
Effects of bivalent cations on adenosine sensitivity in the rat hippocampal slice D.A. Smith
a
and T.V. D u n w i d d i e
b,c
a Neuroscience Program, Oberlin College, Oberlin, OH 44074 (USA), b Department of Pharmacology and Program in Neuroscience, University of Colorado Health Sciences Center, Denver, CO 80262 (USA) and c Veterans Administration Medical Center, Denver, CO 80220 (USA)
(Accepted 15 February 1993)
Key words: Adenosine; A1 receptor; Hippocampus; Ca2+; Mg2+; Presynaptic inhibition; Synaptic modulation
Ligand binding studies have demonstrated that bivalent cations can alter interactions between purinergic agonists and adenosine receptors in brain membranes. In the present study, we have investigated whether a similar interaction can be demonstrated in terms of a functional response to adenosine, which is the inhibition of synaptic transmission in the CA1 region of the rat hippocampus mediated via presynaptic A1 receptors. Our data suggest that alterations in cation concentrations do not significantly affect the sensitivity of these adenosine receptors, as long as changes are made in such a way as to leave presynaptic Ca 2+ entry unaffected. The experimental results do not support the conclusion that there is a specific effect of either Ca z+ or Mg2+ on adenosine receptor sensitivity, such as has been described for agonist interactions with adenosine A1 receptors. We conclude that the effects of bivalent cations observed in ligand binding studies probably reflect an effect at an intracellular site, either on the receptor itself, or perhaps on associated GTP binding proteins.
INTRODUCTION A d e n o s i n e is an important n e u r o m o d u l a t o r 10,26 that both inhibits transmitter release at a variety of central and peripheral synapses 7"14'15A7, and has direct depressant actions on postsynaptic n e u r o n s 19'3°. Both the preand postsynaptic actions of adenosine have b e e n observed in the CA1 region of the hippocampus, a region where adenosine blocks the release of glutamate and hyperpolarizes pyramidal cells 3'4'2°'24'25. T h e s e effects of adenosine are consistent with evidence demonstrating that adenosine A1 receptors are located presynaptically on the axon terminals of CA3 pyramidal cells, and postsynaptically on the dendrites of CA1 pyramidal cells 5. T h e inhibition of transmitter release may involve inhibition of presynaptic Ca 2+ influx 14'22'23, while the postsynaptic effects are primarily related to the activation of a G-protein c o u p l e d potassium channel 29'3°. It is also significant that while adenosine inhibits synaptic transmission at virtually all excitatory synapses in the h i p p o c a m p a l formation, it does not inhibit transmission at G A B A e r g i c synapses is. Thus, the net effect of adenosine in this brain region is to
depress excitatory transmission and reduce postsynaptic excitability, while leaving inhibitory circuits largely unaffected. Because of the p r o f o u n d inhibitory effect that adenosine exerts over electrophysiological activity in the CA1 region, and because there appears to be a continuous basal level of adenosine receptor activation s , it has b e e n suggested that adenosine may be an important tonic regulator of neural activity in the hipp o e a m p u s l°. Conversely, the excitatory effects of methylxanthines such as caffeine and theophylline have b e e n attributed to their ability to block adenosine receptors, and thereby disrupt this tonic inhibitory effect of adenosine 1°'~2. Factors that might regulate this basal activity, such as changes in adenosine receptor number, or changes in the sensitivity of adenosine receptors, could have important effects on electrophysiological activity. In this context, previous reports that bivalent cations such as Ca 2+, Mg 2+, and Mn 2+ can e n h a n c e the specific binding of adenosine receptor agonists (but not antagonists) to adenosine receptors 16'21'31 suggests that this could be a significant m e c h a n i s m by which adenosine sensitivity is regulated.
Correspondence: D. Smith, Neuroscience Program, Sperry Hall, Oberlin College, Oberlin, OH 44074, USA.
62 S o m e p r e v i o u s e l e c t r o p h y s i o l o g i c a l s t u d i e s in t h e hip-
s p i k e is e x t r e m e l y s e n s i t i v e to a g e n t s t h a t c h a n g e c e l l u -
p o c a m p u s h a v e r e a c h e d a s i m i l a r c o n c l u s i o n as well, in
lar excitability, a n d r e s p o n d s in a n o n - l i n e a r f a s h i o n to
t h a t an a p p a r e n t r e d u c t i o n in t h e sensitivity to a d e n o -
c h a n g e s in s y n a p t i c input. F o r t h e s e r e a s o n s , t h e p o p u -
sine is o b s e r v e d
l a t i o n s p i k e is n o t an a c c u r a t e
under
conditions where
the
M g 2+
c o n c e n t r a t i o n in t h e r e c o r d i n g m e d i u m is r e d u c e d or e l i m i n a t e d J,2,27,2~. T h e r e are at least t h r e e p o s s i b l e m e c h a n i s m s t h a t
strength.
indicator of synaptic
T h i s is p a r t i c u l a r l y t r u e in t h e c a s e o f an
a g e n t such as a d e n o s i n e , w h i c h w o u l d b e e x p e c t e d to r e d u c e t h e p o p u l a t i o n spike by v i r t u e o f its ability to
m i g h t e x p l a i n this result. First, as p r o p o s e d by S t o n e
h y p e r p o l a r i z e p y r a m i d a l n e u r o n s , i n d e p e n d e n t l y o f any
a n d c o l l e a g u e s ~, t h e r e m a y be a s p e c i f i c e f f e c t o f M g 2+
e f f e c t s it m i g h t h a v e on s y n a p t i c t r a n s m i s s i o n .
( a n d possibly C o ~+) ions o n a d e n o s i n e
receptors
to
f a c i l i t a t e a d e n o s i n e r e s p o n s e s . A s e c o n d possibility t h a t
MATERIALS AND METHODS
is m o r e in a c c o r d a n c e w i t h t h e l i g a n d b i n d i n g s t u d i e s o f a d e n o s i n e r e c e p t o r s ~6'213~ is t h a t t h e r e is a g e n e r a l ized e f f e c t o f b i v a l e n t c a t i o n s , such t h a t C a " , M g " , o r M n 2+ a n d o t h e r s i m i l a r c a t i o n s can all e n h a n c e a d e n o s i n e action. Finally, as we h a v e p r e v i o u s l y sugg e s t e d 9'~ J, t h e sensitivity o f t r a n s m i t t e r r e l e a s e to m o d u l a t i o n by a d e n o s i n e m i g h t d e p e n d
u p o n the a m o u n t
o f C a 2+ e n t e r i n g t h e n e r v e t e r m i n a l , such t h a t at high C a 2+ c o n c e n t r a t i o n s ,
the effect of adenosine
is re-
d u c e d , w h e r e a s at low C a 2+ c o n c e n t r a t i o n s , t h e e f f e c t o f a d e n o s i n e is e n h a n c e d . In o r d e r
to
determine
which
of these
potential
m e c h a n i s m s is r e s p o n s i b l e for t h e o b s e r v e d e f f e c t s o f b i v a l e n t c a t i o n s on a d e n o s i n e sensitivity, we h a v e c o n ducted
experiments
where
we
have
systematically
c h a n g e d the c o n c e n t r a t i o n s o f C a 2+ a n d M g 2+ in t h e m e d i u m , b u t in such a way t h a t t h e t h r e e p r o p o s e d hypotheses would predict differing effects on adenosine sensitivity. Specifically, we h a v e m a n i p u l a t e d bivalent cation concentrations
in s u c h a w a y t h a t M g 2+
( a n d C a 2+) c o n c e n t r a t i o n s c o u l d b e c h a n g e d w i t h o u t simultaneously
affecting
lease. F u r t h e r m o r e , encountered
basal
neurotransmitter
re-
to a v o i d s o m e o f t h e d i f f i c u l t i e s
in p r e v i o u s s t u d i e s o f b i v a l e n t c a t i o n /
a d e n o s i n e i n t e r a c t i o n s ~'2'27'2s, w e h a v e u s e d t h e f E P S P r e c o r d e d in t h e s t r a t u m r a d i a t u m o f t h e C A 1 r e g i o n as o u r r e s p o n s e m e a s u r e , r a t h e r t h a n t h e p o p u l a t i o n spike response.
It is well e s t a b l i s h e d
that
the population
Transverse, 400 ~m thick, hippocampal slices were obtained from 6- to 8-week-old~ male Sprague-Dawley rats (Sasco Animal Laboratories, Omaha, NE) using methods previously described t3. Briefly, after dissecting the hippocampus from the whole brain, slices were cut with a tissue chopper (Sorvall) and were subsequently transferred to an 0 2/COrartificial cerebral spinal fluid (ACSF) interface holding chamber to equilibrate. The chambers were maintained at 33°C. At least 1 h after surgery, the slices were transferred to a submersion recording chamber ( 1 ml volume) where they were placed on a nylon net and superfused (2 ml/min) with ACSF containing (in raM): NaC1 124. KCI 4.0, KH2PO a 1.0, MgCI.6H20 2.4, CaCIz.2H20 2.5, ~)-glucose 10, and NaHCO 3 26, pH 7.4. The perfusion medium was gassed with humidified 95% 0 2 / 5 % CO 2 and maintained at a temperature of 33-34°C. Extracellular recordings were made using glass microelectrodes (2-4 MO) filled with 2 M NaCI. fEPSPs were recorded by placing the microelectrodes in the stratum radiatum of the CA1 region of the hippocampus. Orthodromic fEPSPs were evoked every 3/1 s using a twisted bipolar nichrome wire electrode which was placed in the stratum radiatum near the border of the CAI and CA2 regions. Stimulation consisted of 0.2 ms square wave pulses. Stimulation voltage was adjusted individually for each slice to produce a fEPSP that was approximately 80% of the maximum response attainable at the start of the experiment. All electrodes were positioned visually. Only slices in which the fEPSP exceeded 1 mV were used for study. Responses were recorded using an AC amplifier, and a computer was used to digitize and store the responses for further analysis. Adenosine hemisulfate (Sigma) dissolved in distilled H 2 0 as a 5 mM stock solution was added to the recording medium via a calibrated syringe pump (Sage). The dose-response relationship for adenosine was determined by exposing slices to incrementally increasing doses (20, 50, 811 and 110 tzM) of adenosine. Each dose was applied in the recording medium for a minimum of 10 min. To assess the effects of reduced Mg 2+ levels on the adenosine dose-response curve, ACSF with the appropriate Mg 2+ and Ca 2+ concentrations was perfused in the recording chamber for at least 111-15 min before assessing the effects of adenosine. The 10-15 min
TABLE 1 The calculated and experimentally determined fEPSP amplitudes for the 4 different types of recording medium used in these experiments
All percentages are related to the amplitude of the response in solution 1 (2.5 mM Ca2+/2.4 mM Mg z+ ). The estimated fEPSP amplitude for each solution was determined as a percent of control using the formula described in the Materials and Methods section. The measured fEPSP amplitudes represent the 80% of maximal response (control ACSF), or for solutions 2-4, the amplitude of the response was set to 80% of maximum in the control ACSF, and the slice was then superfused with one of the alternate buffers and the response amplitude determined once the response had stabilized in the new medium. None of the differences between control and solutions 2-4 were statistically significant. Medium
No. ~)f slices
Ca 2 + conc. (raM)
I (Control) 2 3 4
24 14 16 9
2.5 2.0 1.5 1.5
Mg 2 + conc. (mM)
2.4 1.32 0.25 0
Estimated fEPSP amplitude
JEPSP amplitude (mV)
fEPSP amplitude (percent of control medium)
100% 10(1% 10/)% 115.2%
1.84_+(I.20 1.88 +_(I.28 1.83 + 11.28 2.37+0.31
100 _+10.9% 102.2 _+15.2% 99.5 + 15.2% 128.8_+ 16.8%
63 waiting period was chosen because preliminary observations showed that it took 5-10 min of perfusion with each of the ACSF media before stable changes in fEPSP amplitudes and waveforms were obtained. Typically, for each slice, a dose-response curve was determined in both the standard ACSF (solution 1 below) as well as in at least one of the other types of medium. The order of exposure to different media was varied in a pseudo-random fashion. In total, 4 different ACSF solutions, each with varying concentrations of Mg 2÷ and Ca 2+ were used: (1) 2.5 mM Ca 2+ and 2.4 mM Mg 2÷ (standard ACSF), (2) 2.0 mM Ca 2÷ and 1.32 mM Mg 2+, (3) 1.5 mM Ca 2+ and.25 mM Mg 2+, and (4) nominally Mg2+-free solution containing 1.5 mM Ca 2÷. The concentrations of the other constituents of the ACSF were unaltered from the values used in the standard perfusion medium. The concentrations of Ca 2÷ and Mg 2÷ chosen for all except the Mg2+-free medium were concentrations that should be equieffective in terms of transmitter release, using the relation proposed by Dodge and Rahamimoff 6 for the effects of Ca 2+ and Mg 2+ on EPSP amplitude at the frog neuromuscular junction:
EPSP = K
W [Ca] [Ca] [Mg] 1+ +-K1 K2
where K and W are constants, and K 1 and K 2 are dissociation constants for Ca 2+ and Mg 2+, respectively. As a first approximation, we used the same values for Kj and K 2 determined empirically for the neuromuscular junction (1.1 and 2.97). Preliminary studies indicated that the relationship shown above holds equally well for the hippocampal synapses under study (see Table I).
RESULTS In the present study, we sought to determine whether the sensitivity of field excitatory postsynaptic responses (fEPSPs) to inhibition by adenosine was affected by the concentrations of bivalent cations in the medium. Because it has been demonstrated that the adenosine sensitivity of fEPSP responses is affected by the basal level of neurotransmitter release 9, it was necessary to conduct these studies under conditions where transmitter release was unaffected by the changes in Ca 2+ and Mg 2+ concentrations. The Ca 2+ and Mg 2+ concentrations in solutions 1-3 were estimated to be equieffective in releasing transmitter based upon data from the frog neuromuscular junction, but we wished to demonstrate directly that this was the case in our hippocampal slice experiments. Table I summarizes data comparing the amplitudes of the fEPSPs evoked in the 4 ACSF solutions used in this study. The amplitudes of the fEPSPs evoked in solutions 1-3, which were estimated to have equal Ca 2+ activity, were virtually identical, although the fEPSPs in solutions with lower cation concentration were more likely to show evoked afterdischarges. We were unable to record responses reliably in an equivalent medium that was completely Mg2+-free, because of the extensive spontaneous and evoked bursting that occurred in this medium. Therefore, we used a MgZ+-free buffer with a higher than estimated Ca 2+ concentration (solution 4); as expected,
~
A
~ "~
Ca =* 2.5 Mg =" 2.4
V
ca ~" 1.s M g 2" 0
B Ca 2° 2.5 Mg 2" 2.4
I
1
~
a2" 1.5 Mg2" 0 1 mV 2 msee
Fig. 1. Effects of changing bivalent cation concentrations on evoked responses. The slice illustrated in A was superfused sequentially with 4 different buffers containing the indicated concentrations of Ca 2+ and Mg 2+. As can be seen, there were minor changes in the initial aspects of the response (presynaptic fiber spike and falling phase of the fEPSP), but the longer latency aspects of the waveforms showed marked changes related to firing of the pyramidal neurons. These changes are particularly pronounced in the Mg2+-free medium (bottom record). B: shows recordings from a slice that was switched from the control medium directly to the Mg2*-free buffer; again, while there was relatively little effect on the early part of the response, the later components were markedly affected. Bar = 2 ms and 1 inV.
the fEPSP amplitudes evoked in this buffer were higher than in control slices (Table I). However, under these conditions, the fEPSPs were greatly distorted by repetitive afterdischarges that altered both the shape and the latency of the recorded response (Fig. 1). In such slices, it was impossible to evoke fEPSPs of more than a few hundred microvolts amplitude without eliciting large inverted population spike responses in the dendritic recording. Such inverted population spikes can be observed in slices under control conditions, but only at very high stimulation intensities, and more than one such response is usually not observed. These high amplitude repetitive spikes suggest that the pyramidal neurons are highly excitable in this medium, probably as a result of the loss of the charge-shielding effects of Ca 2+ and Mg 2+ ions bound to the membrane. Because of this distortion, it was difficult to analyze these results in the same manner as the other responses. Clearly, the responses recorded in low or Mg2+-free media are complex and their shapes are distorted, making peak amplitude measures quite unreliable. Accordingly, in the experiments in which the sensitivity to adenosine was assessed, we also measured the initial slope of the fEPSP, an aspect of the fEPSP unlikely to
64 be contaminated by afterdischarges (see below). It was also apparent in many (Fig. 1A) but not all (Fig. 1B) slices, that the amplitude of the presynaptic fiber spike increased in amplitude as Mg 2+ and Ca 2+ concentrations were reduced. However, we did not observe any systematic differences in adenosine sensitivity between slices in which the fiber spike was increased in low Mg 2+ and Ca 2+ conditions, and those slices in which this effect was not observed. Fig. 2 presents representative examples of fEPSPs recorded in medium containing 2.5 mM Ca 2+/2.4 mM Mg 2+, and in the Mg2+-free ACSF, and illustrates the effects of adenosine on these responses. The examples recorded in standard ACSF illustrate the fact that both the amplitude of the fEPSP and the initial slope of this response were inhibited in a concentration-specific manner by adenosine superfusion. In the slices in
Mg2+-free medium, adenosine also inhibited the response; this was readily apparent in terms of the initial slope of this potential, but changes in the maximum amplitude of the fEPSP response could not be determined reliably because of the effects of inverted population spike responses. In some of the slices maintained in Mg2+-free medium, it appeared that low concentrations of adenosine actually increased the inverted population spike responses, while higher concentrations invariably abolished this component of the response. The effects of adenosine on fEPSP amplitude in the 3 ACSF solutions with approximately equivalent effective calcium concentrations are summarized in Fig. 3. In each case, adenosine reduced the peak amplitude of the fEPSP significantly (F2,.~ ~ = 241, P < 0.001) and in a concentration-dependent manner. However, the depressant effects of adenosine on fEPSP amplitude did
C
A
110 uM 110 uM 80 uM 50 uM 20 uM
80 uM 50 uM 20 uM
Control Control
D
B
110 uM 110 uM 80 uM
80 uM
50 uM
50 uM
20 uM Control
20 uM Control
Fig. 2. Effects of adenosine on field potentials in control medium, and in medium containing 1.5 m M Ca 2+ and 0 mM Mg 2+. The lower 5 records in each panel correspond to averaged responses recorded during superfusion with 110, 80, 50, 20, and 0 txM adenosine, whereas the upper record shows the same 5 averages superimposed. A and B are from slices maintained in control medium; in responses that were not contaminated by inverted population spikes (as in A), the EPSP could be measured by determining the peak amplitude of the response (indicated by vertical line), whereas in slices in which an inverted spike was observed (seen as a positive-going potential superimposed upon the negative fEPSP in B), the response was measured on the initial aspect of the fEPSP. T h e vertical line superimposed on the records in B through D indicates the time point at which m e a s u r e m e n t s of fEPSP slope were made. C and D illustrate responses evoked in Mg2+-free medium, in which multiple positive-going peaks were superimposed upon the fEPSP response. The records in B and D were obtained from the same slice but in different media. The ICs~~ values determined for these slices for adenosine were 38 txM, 41 p~M, 77 ~ M , and 36 txM for the slices shown in A - D , respectively.
65 lOO
~.
,m
/zM, and 44 + 3.7/zM in buffer solutions 1-4, respectively.
so
CI2.5/Mg 2.4 Ca2.01Mg1.32
eo
~g
,o
o uu
20
o
20 uM
50 uM 80 uM ADENOSINE CONCENTRATION
110 uM
Fig. 3. Effect of adenosine on peak fEPSP amplitude. The adenosine-mediated depression of fEPSP amplitude (mean+S.E.M.) is shown for media 1-3, which were approximately equivalent in supporting neurotransmitter release. The fEPSP amplitudes were measured as the peak negativity whenever possible under each condition. Every concentration of adenosine tested had essentially the same effects on the fEPSP response in each of the buffers.
not differ between the 3 media ( F 6 , 1 4 3 = 0.479, P < 0.822). Because of the difficulties in measuring the fEPSP in Mg2+-free buffer, we reanalyzed all of the experiments by measuring the slope of the early initial phase of the EPSP response, before any distortion induced by firing of the CA1 pyramidal neurons. Conc e n t r a t i o n - r e s p o n s e curves for adenosine under each of these conditions are shown in Fig. 4. Statistical analysis of these data reveals no concentration × ACSF condition interaction (F3,14 4 = 0.913, P < 0.515). The IC50 values were 42 + 3.2 /xM, 40 + 3.6 /xM, 36 + 2.7
1 O0 IJJ (2.
q
.-o--
Ca2.5/Mg2.4
•- e - -
Ca2.0/Mgl.3
8o
w z "3 w
60
,n
40
z uJ 0 w a.
20
DISCUSSION
Ca1.51Mg.25
T
.-O-.- C a 1 . 5 / M g . 2 5 .5/MgO
20 ADENOSINE
50 CONCENTRATION
100 (uM)
Fig. 4. Dose-response curves for adenosine. Log dose-response curves are shown for adenosine-mediated depression of the fEPSP in each of the buffers tested; the measurement in each case was the slope of the fEPSP, not the maximum amplitude. Each point represents the mean _+S.E.M. for 24 (2.5 mM Ca2+/2.4 mM Mg2+), 14 (2.0 mM Ca2+/1.32 mM Mg2+, 16 (1.5 mM Ca2+/.25 mM Mg2+), and 9 (1.5 mM Ca2+/0.0 mM Mg2+ slices tested with the indicated concentration of adenosine. IC50 values for the 4 conditions were 42 /zM, 40/~M, 36/xM, and 44 #M, respectively. There was no overall effect of buffer composition on the IC50 values determined in this manner. A few of the individual points in the display were offset slightly to enhance clarity.
Biochemical studies have demonstrated that bivalent cations can modify the interactions between agonist ligands and G-protein-coupled transmitter receptors. However, there are few functional studies that would support the hypothesis that cations are physiologically relevant modifiers of receptor sensitivity. We have examined whether the modulation of neurotransmitter release mediated via hippocampal adenosine A1 receptors shows this type of cation sensitivity. However, these experiments are complicated by the fact that the response that is measured (transmitter release), is itself a Ca2+-dependent process, and will be changed by alterations in bivalent cation concentrations. Therefore, we have developed and tested three not necessarily exclusive hypotheses that could account for bivalent cation effects on adenosine sensitivity. The first hypothesis is that there is a Mg2÷-specific effect on adenosine receptors, such that both ligand binding and functional responses are enhanced by Mg 2+. A second hypothesis, which is perhaps more in accord with the data from radioligand binding studies 16'21'31, is that a variety of bivalent cations, including Ca e ÷, MgZ ÷, Mn z +, Co 2÷, Sr 2÷, and Ba e+, can all enhance adenosine binding and functional responses, albeit with somewhat different potencies and efficacies. A third alternative, which we have proposed based upon our previous electrophysiological studies, is that the sensitivity of neurotransmitter release to inhibition by adenosine is dependent upon the amount of Ca 2+ entering the nerve terminal; at low levels of Ca 2÷ entry, adenosine is more potent in inhibiting release, whereas its potency drops with higher levels of Ca 2÷ influx. Differentiating between these alternative hypotheses is somewhat difficult, because every bivalent cation that has been demonstrated to modulate agonist binding to adenosine receptors also competes with Ca 2÷ for the presynaptic Ca 2÷ channels that mediate neurotransmitter release. For this reason, it has been necessary to develop protocols whereby the effects of cations on neurotransmitter release per se can be distinguished from possible effects on adenosine receptors. The results of the present investigations would clearly seem to rule out the first hypothesis, viz., that there is a Mg2+-specific effect on adenosine A1 receptor-mediated responses. In these experiments, we have altered the Mg 2÷ concentration in the medium by a factor of almost 10, with no detectable effect on the potency of adenosine in inhibiting synaptic transmis-
66 sion. Although there are technical difficulties involved in recording from slices in nominally Mg2+-free medium, it appeared that even under these conditions, adenosine sensitivity was not markedly altered. Whether the fEPSP slope or amplitude was used as the measure of adenosine's effect, synaptic responses were essentially abolished with the same concentration of adenosine regardless of the Mg 2+ concentration, supporting the conclusion that the effects of adenosine are not markedly attenuated by reduced Mg 2+. The second hypothesis (that the total concentration of bivalent cations regulates adenosine sensitivity) would also seem to be ruled out by these experiments as well, because changing from medium containing a total of 4.9 mM of bivalent cations to 1.75 mM had no significant effect upon sensitivity. Furthermore, in a previous study 9 we have demonstrated that increasing the Ca 2+ concentration of the medium from 1.0 to 10 mM (with Mg 2+ fixed at 2.4 raM) actually decreases adenosine sensitivity, the opposite of what would be predicted by the second hypothesis. Thus, the present findings would not seem to support previous studies suggesting that bivalent cations modify agonist interactions with adenosine A1 receptors. However, it should be stressed that there is little evidence for a bivalent cation effect on adenosine receptors in intact systems; the evidence upon which these hypotheses are based comes primarily from studies of adenylyl cyclase and ligand binding, which are conducted in membrane preparations, not in intact cells. Furthermore, there is considerable evidence to suggest that Mg 2+ effects may occur as a result of interactions with GTP-binding proteins 3~, which are thought to be intracellular, not extracellular proteins. For these reasons, we conclude that the bivalent cation effects that have been described in binding and cyclase studies probably reflect an interaction with an intracellular site, and that the present studies have not revealed effects upon adenosine sensitivity because changing the cation concentrations in the extracellular buffer has little effect upon the intracellular concentrations. As far as the third hypothesis is concerned, i.e. that adenosine sensitivity is inversely related to the amount of Ca 2+ entering the nerve terminal, the present results are consistent with predictions based upon this hypothesis. This hypothesis would suggest that if the bivalent cation concentrations are manipulated so that there is no net effect of changes in Mg 2+ and Ca 2+ concentrations on transmitter release, then no changes would be expected in adenosine sensitivity, and this was in fact the result that was observed. In the one situation where transmitter release was not equivalent (in Mg2+-free medium), the synaptic response was
somewhat greater, and the modulatory effect of adenosine correspondingly reduced, although this effect was small. This type of action was demonstrated more clearly in a previous study, where manipulations of Ca :+ and Mg 2+ that resulted in changes in the basal release of transmitter-produced highly significant changes in the adenosine sensitivity of the response 9. The basis for the Ca 2+ sensitivity of the adenosine response is not clear, but might reflect the intrinsic Ca ~+ dependency of the release process. At low levels of extracellular Ca z+, the release of transmitter varies as a 4th order function of the Ca z+ concentration, which means that very small changes in Ca 2+ have highly significant effects upon release, whereas at higher concentrations, the release process appears to saturate, such that increases in Ca z+ have less and less additional effect. If adenosine modulates the activity of a presynaptic Ca 2+ channel, or the Ca 2+ sensitivity of some aspect of the release process, then its effects should be much greater at lower rather than higher levels of Ca z+. An alternative explanation based upon the consequences of non-linear summation of EPSPs at these synapses has also been proposed (see ref. 9 for a discussion of this possibility), and would also be in accordance with the observations in the present study. One report whose results initially seem inconsistent with the general conclusions reached in this study is that of Bartrup and StoneJ; this study reported that reducing the Mg z+ concentration of the medium appeared to reduce the adenosine sensitivity of evoked population spikes in hippocampus. However, there are several aspects of this study that make this conclusion equivocal. First, these experiments assessed the effect of changing Mg 2+ on presynaptic adenosine receptors not by measuring the amplitude of the fEPSP response, which provides a direct measure of synaptic current, but rather by measuring the population spike produced by stimulation of the excitatory inputs to the pyramidal neurons. Because the population spike reflects postsynaptic cell firing, it is impossible to determine whether the changes produced by reducing Mg 2+ reflect increased cellular excitability resulting from changes in bivalent cation concentration, changes in postsynaptic adenosine receptor sensitivity, or changes in presynaptic adenosine sensitivity. The initial slope of the fEPSP response would be sensitive to only the last of these three actions, and is the only direct means to demonstrate changes in presynaptic adenosine receptor sensitivity unconfounded by other effects of bivalent cations. Because the population spike was used as the response measure in Bartrup and Stone's study, this also meant that it was not possible to adequately control for the changes in EPSP amplitude induced by changing the
67
Mg 2+ concentration, because the EPSP was never directly measured; normalizing the population spike response by reducing Ca 2+ does not accomplish this, because the population spike response reflects not only the strength of the synaptic input, but also changes in pyramidal neuron excitability. In addition, this study only investigated the effects of reductions in Mg 2+ concentration on adenosine sensitivity, whereas the binding data clearly indicate that Ca 2+ is, if anything, even more effective than Mg 2+ in enhancing adenosine receptor binding; if this were the case, then one would predict that reducing Ca 2+ concentrations should also reduce adenosine sensitivity, and in fact the opposite result appears to be the case 9. In summary, we conclude that most if not all of the available data suggest that the sensitivity of presynaptic adenosine receptors to adenosine in the CA1 region of the hippocampus is unaffected by changes in Mg 2+ concentrations. However, any manipulation that alters the Ca 2+ influx into the presynaptic nerve terminal (and correspondingly alters basal transmitter release as well), alters the apparent sensitivity of the release process to inhibition by adenosine. This effect is observed not only at high levels of Ca z+ entry, where the release mechanism might be 'saturated' with Ca 2+, but at lower levels of release as well. The functional significance of the effects of bivalent cations on adenosine receptor binding are currently unclear. If, as we hypothesize, this reflects the actions of cations at an intracellular site, and possibly on G-proteins rather than the receptor itself, then this might provide an intracellular site at which the sensitivity of the adenosine system could be regulated. Further studies will be required to demonstrate whether or not this is the case. Acknowledgements. This research was supported by Grant NS29173 (T.V.D.) and the VA Medical Research Service.
REFERENCES 1 Bartrup, J.T. and Stone, T.W., Interactions of adenosine and magnesium on rat hippocampal slices, Brain Res., 463 (1988) 374-379. 2 Bartrup, J.T. and Stone, T.W., Presynaptic actions of adenosine are magnesium-dependent, Neuropharmacology, 27 (1988) 761763. 3 Burke, S.P. and Nadler, J.V., Regulation of glutamate and aspartate release from slices of the hippocampal CA1 area: effects of adenosine and baclofen, J. Neurochem., 51 (1988) 1541-1551. 4 Corradetti, R., Lo Conte, G., Moroni, F., Passani, M.B. and Pepeu, G., Adenosine decreases aspartate and glutamate release from rat hippocampal slices, Eur. J. Pharmacol., 104 (1984) 19-26. 5 Deckert, J. and Jorgensen, M.B., Evidence for pre- and postsynaptic localization of adenosine A1 receptors in the CA1 region of the rat hippocampus: a quantitative autoradiographic study, Brain Res., 446 (1988) 161-164.
6 Dodge, F.A. and Rahamimoff, R., Co-operative action of calcium ions in transmitter release at the neuromuscular junction, J. Physiol., 193 (1967) 419-432. 7 Dolphin, A.C. and Archer, E.R., An adenosine agonist inhibits and a cyclic AMP analogue enhances the release of glutamate but not GABA from slices of rat dentate gyrus, Nature, 43 (1983) 49-54. 8 Dunwiddie, T.V., Endogenously released adenosine regulates excitability in the in vitro hippocampus, Epilepsia, 21 (1980) 541-548. 9 Dunwiddie, T.V., Interactions between the effects of adenosine and calcium on synaptic responses in rat hippocampus in vitro, J. Physiol., 350 (1984) 545-559. 10 Dunwiddie, T.V., The physiological role of adenosine in the central nervous system, Int. Rev. Neurobiol., 27 (1985) 63-139. 11 Dunwiddie, T.V. and Haas, H.L., Adenosine increases synaptic facilitation in the in vitro rat hippocampus: evidence for a presynaptic site of action, J. Physiol., 369 (1985) 365-377. 12 Dunwiddie, T.V., Hoffer, B.J. and Fredholm, B.B., Alkylxanthines elevate hippocampal excitability: evidence for a role of endogenous adenosine, Naunyn Schmiedebergs Arch. Pharmacol., 316 (1981) 326-330. 13 Dunwiddic, T.V. and Lynch, G.S., Long-term potentiation and depression of synaptic responses in the rat hippocampus: localization and frequency dependency, J. Physiol., 276 (1978) 353-367. 14 Fredholm, B.B. and Dunwiddie, T.V., How does adenosine inhibit transmitter release?, Trends Pharmacol., 9 (1988) 130-134. 15 Fredholm, B.B. and Hedqvist, P., Modulation of neurotransmission by purine nucleotides and nucleosides, Biochem. Pharmacol., 29 (1980) 1635-1643. 16 Goodman, R.R., Cooper, M.J., Gavish, M. and Snyder, S.H., Guanine nucleotide and cation regulation of the binding of [3H]cyclohexyladenosine and [3H]diethylphenylxanthine to adenosine A1 receptors in brain membranes, Mol. Pharmacol., 21 (1982) 329-335. 17 Harms, H.H., Wardeh, G. and Mulder, A.H., Adenosine modulates depolarization induced release of 3H-noradrenaline from slices of rat brain neocortex, Eur. J. Pharmacol., 49 (1978) 305308. 18 Lambert, N.A. and Teyler, T.J., Adenosine selectively depresses excitatory synaptic transmission in area CA1 of the rat hippocampus, Neurosci. Lett., 122 (1991) 50-52. 19 Lin, Y. and Phillis, J.W., Characterization of the depression of rat cerebral cortical neurons by selective adenosine agonists, Brain Res., 540 (1991) 307-310. 20 Proctor, W.R. and Dunwiddie, T.V., Adenosine inhibits calcium spikes in hippocampal pyramidal neurons in vitro, Neurosci. Lett., 35 (1983) 197-201. 21 Reddington, M., Alexander, S.P., Erfurth, A., Lee, K.S. and Kreutzberg, G.W., Biochemical and autoradiographic approaches to the characterization of adenosine receptors in brain. In E. Gerlach and B.F. Becker (Eds.), Topics and Perspectiues in Adenosine Research, Springer, Berlin, 1987, pp. 49-58. 22 Scholz, K.P. and Miller, R.J., Analysis of adenosine actions on calcium currents and synaptic transmission in cultured rat hippocampal pyramidal neurones, J. Physiol., 435 (1991) 373-393. 23 Schubert, P., Heinemann, U. and Lee, K.S., Differential effect of adenosine on pre- and postsynaptic calcium fluxes, Brain Res., 376 (1986) 382-386. 24 Segal, M., Intracellular analysis of a postsynaptic actions of adenosine in the rat hippocampus, Eur. J. Pharmacol., 79 (1982) 193-199. 25 Siggins, G.R. and Schubert, P., Adenosine depression of hippocampal neurons in vitro: an intracellular study of dose-dependent actions on synaptic and membrane potentials, Neurosci. Lett., 23 (1981) 55-60. 26 Stone, T.W., Adenosine in the Nervous System, Academic Press, San Diego, 1991. 27 Stone, T.W. and Bartrup, J.T., Magnesium-dependency of presynaptic inhibition by adenosine. In J.A. Ribeiro (Ed.), Adenosine Receptors in the Neruous System, Taylor and Francis, London, 1989, pp. 87-94.
68 28 Stone, T.W., Connick, J.H. and Bartrup, J.T., NMDA-receptorindependent effects of low magnesium: involvement of adenosine, Brain Res., 508 (1990) 333-336. 29 Trussell, L.O. and Jackson, M.B., Adenosine-activated potassium conductance in cultured striatal neurons, Proc. Natl. Acad. Sci. USA, 82 (1985) 4857-4861.
30 Trussell, L.O. and Jackson, M.B., Dependence of an adenosineactivated potassium current on a GTP-binding protein in mammalian central neurons, J. Neurosci., 7 (1987) 3306-3316. 31 Yeung, S.M., Fossom, L.H., Gill, D.L. and Cooper, D.M., Magnesium ion exerts a central role in the regulation of inhibitory adenosine receptors, Biochem. J., 229 (1985) 91-100.