Adenosine A1 Receptors Mediate Retinotectal Presynaptic Inhibition: Uncoupling by C-Kinase and Role in LTP During Regeneration

Chunyi Zhang and John T. Schmidt

Department of Biological Sciences and Neurobiology Research Center, State University of New York, Albany, New York 12222

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Zhang, Chunyi and John T. Schmidt. Adenosine A1 receptors mediate retinotectal presynaptic inhibition: uncoupling by C-kinase and role in LTP during regeneration. J. Neurophysiol. 79: 501-510, 1998. Presynaptic adenosine receptors inhibit transmitter release at many synapses and are known to exist on retinotectal terminals. In this paper we show that adenosine decreases retinotectal field potentials by ~30% and investigate the mechanism. First, as judged by the effects of specific calcium channel blockers, retinotectal transmission was mediated almost exclusively by N-type calcium channels, which are known to be modulated by adenosine A1 receptors. Transmission was completely blocked by either omega -Conotoxin GVIA (-100%, N-type blocker) or omega -Conotoxin MVIIC (-99%, N-, P- and Q-type blocker) and was not significantly affected by omega -Agatoxin IVA [+1.7 ± 9.3% (SE), P-,Q-type blocker], but was augmented slightly by nifedipine(+9.3 ± 2.1%, L-type blocker). Second, the adenosine inhibition was presynaptic, as indicated by a 43% increase in paired-pulse facilitation. Third, the selective A1 agonist cyclohexyl adenosine (CHA) at 50 nM caused a 21% decrease in amplitude and the selective A2 agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) at 100 nM caused a 24% increase. Fourth, the selective A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) alone produced an increase in the field potential, suggesting a tonic inhibition mediated by endogenous adenosine. Fifth, pertussis toxin eliminated adenosine inhibition implicating Gi or Go protein coupling. Sixth, C-kinase activation eliminated the A1-mediated inhibition. In regenerating projections, adenosine also caused a decrease in transmission (-30 ± 12%), but after induction of long-term potentiation (LTP) via trains of stimuli or via treatment with the phosphatase inhibitor okadaic acid, the adenosine response was converted to an augmentation. Because LTP is associated with C-kinase activation, this is consistent with C-kinase uncoupling the A1 receptor from inhibiting N-type Ca2+ channels. This uncovers the A2-mediated augmentation as demonstrated in normals with DPMA. Such an effect could account in part for the LTP of immature synapses and the change from rapidly fatiguing to robust synaptic transmission.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Adenosine is a major inhibitory modulator in the CNS that plays a role in a variety of processes, including sleep, anxiety, protection from excess glutamate release (and its ensuing toxicity) and more subtle synaptic modulation (Williams 1987). The inhibitory modulation is mediated by the A1 receptor and can be either presynaptic or postsynaptic (Dolphin 1995; Fredholm and Dunwiddie 1988). The A2 receptor, in contrast, augments synaptic responses.

Adenosine inhibits neurotransmitter release from presynaptic terminals via a pertussis toxin-sensitive G-protein (Dolphin 1995; Dolphin and Prestwich 1985). The simultaneous measurement of Ca2+ in terminals shows that this inhibition is mediated by a decrease in Ca2+ current, primarily through N-type Ca2+ channels (Dittman and Regehr 1996; Mogul et al. 1993; Yawo and Chuhma 1993). The adenosine A1 receptor is probably directly coupled to the Ca2+ channels through a G-protein, as has been seen with other neurotransmitters exerting similar effects (Clapham 1994; Dolphin 1995; Swartz et al. 1993). This inhibition of N-type Ca2+ channels can be interrupted by activation of protein kinase C (Barrie and Nicholls 1993; Budd and Nicholls 1995; Sanchez-Prieto et al. 1996), one target of which is the pertussis toxin-sensitive G-protein (Katada et al. 1985). Thus adenosine provides an important modulatory system in the CNS with further links to other modulatory second messenger pathways.

In the visual system, the retinal ganglion cells contain adenosine and have A1 receptors on their surfaces, including on their axons (Blazynski et al. 1989; Braas et al. 1987). Adenosine A1 (but not A2) receptors in optic tectum are located on the retinal terminals (Goodman et al. 1983; Wan and Gieger 1990). Although adenosine has been shown to inhibit transmitter release at many different synapses (Barrie and Nicholls 1993; Prince and Stevens 1992; Yawo and Chuhma 1993), adenosine inhibition has not been demonstrated at the retinotectal synapse. Thus in the present study, we investigated the effect of adenosine on synaptic transmission at the goldfish retinotectal synapse, by using a convenient in vitro preparation (King and Schmidt 1991b). The results presented here show that presynaptic release is inhibited by adenosine A1 receptors acting through pertussis toxin (PTX)-sensitive G-proteins, presumably by inhibiting the N-type Ca2+ channel that we show mediate retinotectal transmitter release and that C-kinase activation interrupts this inhibition.

In addition, we also investigated the role of this adenosine inhibition in the immature regenerating projection. Regenerating optic fibers reach the tectum at ~2 wk, begin to establish measurable transmission at around day 18, and go through a sensitive period for activity-driven retinotopic sharpening for several months after this point (Eisele and Schmidt 1988; Schmidt et al. 1983). Our previous studies have shown that responses in immature projections fluctuate greatly and fatigue extremely rapidly (Schmidt et al. 1983). After induction of long-term potentiation (LTP) in these projections, however, the response is not only far larger but also very stable and resistant to fatigue (Schmidt 1990). A number of observations suggested that adenosine inhibition may play a role in this alteration of response properties: 1) the fluctuating response is likely to be the result of a fluctuation of presynaptic transmitter release, 2) immature synapses are likely to have less effective uptake mechanisms for adenosine making them more susceptible to its inhibition, and 3) LTP induction requires C-kinase activation, which might then uncouple the adenosine inhibition. As expected, LTP induction in regenerating projections interrupts this presynaptic adenosine inhibition, uncovers an adenosine A2 receptor mediated augmentation as in normals and alters the synaptic response characteristics in ways that facilitate further stabilization.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Recording of field potentials

Experiments were carried out in goldfish (Carassius auratus). Each fish was anesthetized by immersion in ice water and the tectum was dissected together with the optic tract in ice-cold Ringer solution. After dissection, the tectum and the attached nerves were placed in a recording chamber and continuously superfused with oxygenated Ringer solution at a rate of ~2 ml/min. The temperature of the Ringer solution was adjusted to 13.6-13.9°C. The optic nerve was supramaximally stimulated with a bipolar suction electrode and an isolated stimulator. The evoked field potentials were recorded with a glass micropipette. The signal was amplified, digitized, and stored in a 386 computer with a Data Translation 2801A A/D board and software written by John Dempster (available from Dagan, Minneapolis, MN).

Application of drugs

Drugs were dissolved in appropriate solvents and stored as frozen aliquots. At the time of application, an aliquot was diluted in Ringer solution to the desired concentration and was applied by superfusion. Preliminary experiments have shown that it takes about 15 min for the applied drug to reach a plateau of action. Thus in all experiments the effect of the tested drug was measured as the change in the amplitude of the major negative potential recorded between 15 and 30 min after starting the application of the drug compared with the control value obtained as described below. In the cases where the effect of phorbol ester or receptor antagonist was studied, they were applied starting 30 min before and continued during the application of the other drugs, on which the influence of the phorbol ester or receptor antagonist was tested.

In two other series, we tested the effect of calcium channel blockers on transmission or of pertussis toxin on the inhibitory action of adenosine and N6-cyclohexyladenosine (CHA). In these experiments the preparation was incubated on ice for 1 h with omega -Conotoxin (Ctx) GVIA, omega -Conotoxin MVIIC, nifedipine, or omega -Agatoxin (AgaTx) IVA, or for 2.5-3 h with pertussis toxin (1 µg/ml) in preoxygenated Ringers. Previous tests showed that this period at 0°C had no effect on the nature or amplitude of the responses later recorded. For the Ca2+ channel blockers, responses were then recorded at four to five different positions within both the treated and the control opposite tectum and averaged for comparison. This conserved expensive peptide and pertussis toxins.

Crush of optic nerve

After anaesthesia by immersion in a 0.1% solution of tricaine methanesulfonate (TMS, Crescent Res. Chem., Paradise Valley, AR), the optic nerve was exposed by an incision over the eye and crushed under visual control by using fine curved forceps (Schmidt 1990). Fish were then revived and housed at 20-21°C until the retinotectal projections were used in experiments on regenerating projections paralleling those on normal projections.

Data analysis

Unless otherwise stated, five field potentials elicited by stimulation of the optic tract at a frequency of 0.2 Hz were collected and averaged together. The amplitude of the major negative component was measured as control for testing the effect of drugs. In cases in which the amplitude after drug washout did not recover completely to control levels, five postdrug potentials were collected and averaged with the predrug level and used as the control for evaluating the drug action. Comparison of means was performed with either Student's t-test or one-way analysis of variance(ANOVA) as appropriate. Numerical data reported in the text aremean ± SE. A difference with P value <0.05 was regarded as statistically significant.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Nature of the field potentials.

Stimulation of the optic tract elicited a field potential (e.g., Fig. 1) that consisted of several components: a presynaptic action-potential volley, followed immediately by a monosynaptic response and a later, more variable, polysynaptic component caused by recurrent excitation (King and Schmidt 1991b). The shapes of the synaptic potentials were dependent on the depth of the recording electrode, negative in superficial tectum and inverting to positive in deeper tectum (Schmidt 1979). In the present experiments we consistently placed the recording electrode tip in the retinal synaptic layer at the depth of about 100 µm, where the early negative component reflecting monosynaptic transmission was maximum in amplitude. All changes reported in the following sections are measured as the changes in the amplitude of this negative component.


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FIG. 1. Effect of specific calcium-channel blockers on retinotectal synaptic transmission. Traces are averages of 10 responses recorded in retinal synaptic layer at a depth of 100 µm after supramaximal optic tract stimulation at 0.1 Hz. Beginning of each trace includes a calibration pulse of 1 mV and 2 ms. up-arrow , time of stimulation (artifact electronically suppressed). Response consists of a short-latency presynaptic volley followed immediately by a large negative monosynaptic response and a later, smaller negative polysynaptic response that is more variable. Response after 1 h treatment with each blocker is superimposed on control taken either from same tectum before treatment (A) or from opposite tectum from same animal (B, C, and D). Toxins were applied at following concentrations: Nifedipine, 50 µM; omega -conotoxin (Ctx) GVIA, 2 µM; omega -agatoxin (Agatx) IVA, 0.2 µM; omega -Ctx MVIIC, 1 µM. Complete block of transmission (B and D) uncovers a small positive-going wave that is the result of antidromic activation of tectal cells that give rise to efferents projecting to retina (Schmidt 1979).

N-type Ca2+ channels mediate synaptic transmission

Because adenosine is known to modulate N-type Ca2+ channels (Dolphin et al. 1995; Yawo and Chuhma 1993), we applied specific blockers of Ca2+ channels to determine which types mediate transmitter release from retinal terminals. The results are shown in Fig. 1. First, nifedipine (10 to 50 µM), a dihydropyrridine and L-specific blocker, consistently failed to block any portion of the monosynaptic response, and actually resulted in a slight but consistent augmentation (+9.4 ± 2.1%; n = 3). In contrast, the N-type blocker, omega -Ctx GVIA at 1-2 µM, consistently blocked 100% of the response amplitude (6 cases). The transmission did not recover at all even after 8 h and after several strong tetani (no posttetanic potentiation). The funnel web spider toxin, omega -AgaTx IVA (0.2 µM), which is known to block P-type channels at nanomolar and Q-type channels below micromolar concentrations, did not consistently decrease responses, which averaged 101.7 ± 9.3% of normal (5 cases). Finally, omega -Ctx MVIIC (1 µM), which blocks N-, P-, and Q-type Ca2+ channels, also blocked all of the response amplitude (-98.8 ± 1.2%; n = 4). In each case in which the monosynaptic component was blocked, the presynaptic volley was generally unchanged. The simplest interpretation is that transmitter release at the retinotectal synapse is predominantly dependent on calcium entry through N-type Ca2+ channels, the type most readily modulated by adenosine elsewhere (Yawo and Chuhma 1993).

Adenosine-inhibited transmission

Exogenous adenosine inhibited the field potentials evoked by optic nerve stimulation in a manner that was concentration-dependent in the range of 5-100 µM. At 100 µM, adenosine decreased the amplitude of the field potential by 30.3 ± 1.5% (n = 8, P < 0.001). Figure 2A shows an example of the concentration-dependence of the inhibition and Fig. 2B shows the pooled data. The inhibition produced by adenosine was completely reversible; after the preparation was returned to normal Ringer, the amplitude of the field potential recovered to its control level within 15 min.


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FIG. 2. Adenosine inhibits retinotectal transmission in a concentration-dependent manner. Adenosine was superfused starting at lowest concentration. When effect of each concentration reached a plateau (15-30 min), five traces were taken and next higher concentration was applied. A shows sample records obtained from one preparation. Conventions are as in Fig. 1. B: pooled data from 3 experiments of this kind. Each point is significantly different from its neighboring points (up to 50 µM) and shows progressively larger inhibition produced by increasing adenosine concentrations between 5 and 100 µM.

Adenosine inhibition was mediated by A1 receptors

The two major subtypes of adenosine receptors, A1 and A2, have different pharmacological profiles and signal transduction mechanisms. We investigated which receptor subtype might mediate the adenosine inhibition by using 2 approaches. First, we tested whether or not A1 or A2 agonists could mimic the inhibition produced by adenosine. As shown in Fig. 3, the selective A1 receptor agonist N6-cyclohexyladenosine (CHA) (Bruns et al. 1980) produced inhibition similar to that of adenosine. At 50 nM, CHA inhibited the field potential by 21.13 ± 1.75% (n = 7, P < 0.001; Compare Fig. 3, A and B). In contrast, the selective A2 agonistN6 - [ 2 - ( 3 , 5 - dimethoxyphenyl ) - 2 - ( 2 - methylphenyl ) - ethyl ]adenosine (DPMA; Bridges 1989) not only did not inhibit, but actually produced a significant augmentation of the field potentials (Fig. 3D). At 100 nM, the increase averaged22.4 ± 3% (n = 8, P < 0.01). These results indicate that the adenosine inhibition is most likely mediated by A1 receptors. Second, we showed that the selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; Lohse et al. 1987) applied at 100 nM largely eliminated the adenosine inhibition (Fig. 3C). The inhibition produced by adenosine (100 µM) in the presence of DPCPX was only 3.4 ± 1.2% of control (n = 3), significantly smaller than inhibition in the absence of the antagonist (30.3 ± 1.5%, P < 0.05). Together, these experiments demonstrate that the adenosine inhibition is mediated by the A1 subtype of adenosine receptors.


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FIG. 3. Adenosine inhibition is mediated by adenosine A1 receptors. A: sample record showing inhibition produced by adenosine (100 µM). B: similar inhibition produced by selective A1 receptor agonist N6-cyclohexyladenosine (CHA, 50 nM; there was no consistent effect on polysynaptic component, which was highly variable). C: inhibition produced by adenosine was completely antagonized by selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) at concentration of 100 nM. D: selective A2 receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA;100 nM) did not produce inhibition but rather an augmentation of field potential. These results demonstrate that inhibition produced by adenosine is mediated by adenosine receptors of A1 subtype.

Adenosine inhibition was presynaptic

At central synapses, adenosine could inhibit synaptic transmission by either presynaptic or postsynaptic mechanisms (or both). We tested whether or not adenosine inhibits neural transmission mainly by acting on presynaptic receptors, by using the paired-pulse facilitation test. At normal synapses, when two stimulus pulses are paired at intervals of 40-60 ms, the signal evoked by the second pulse is not greatly augmented by the residual Ca2+ from the first, presumably because the first pulse decreases the number of releasable vesicles because of the high probability of release at each synaptic bouton. Presynaptic inhibition would lower the probability of release so that the effect of the residual Ca2+ in the synaptic terminals becomes much larger. Hence the second signal is often markedly increased in size. To prevent postsynaptic activation of inhibitory circuits, which could interfere with the test, we decreased the intensity of stimulation to keep the field potentials below 1.5 mV. Under such conditions, the paired-pulse facilitation should be a reliable test for presynaptic change during adenosine inhibition (Schulz et al. 1994; Wheeler et al. 1994; Wu and Saggau 1994). Figure 4 shows examples in which adenosine (100 µM) and CHA (50 nM) substantially increased paired-pulse facilitation over that seen before application. The increases in paired-pulse facilitation produced by CHA (50 nM) and adenosine (100 µM) were similar in magnitude and the data were pooled. Overall, in 11 control preparations, the amplitude of the field potential evoked by the second pulse was 132 ± 14% of the first. In the presence of adenosine or CHA, this paired-pulse facilitation was increased to 191 ± 18% (n = 11), which was significantly greater (P < 0.001). In contrast, the A2 agonist DPMA had no effect on paired-pulse facilitation. The paired-pulse facilitation was 103 ± 10% and 98 ± 2% in the absence and presence, respectively, of 100 nM DPMA, (n = 3, P > 0.05). These data demonstrate that the major mechanism of adenosine inhibition is presynaptic although a minor postsynaptic component cannot be ruled out from this data.


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FIG. 4. Adenosine and CHA inhibition is associated with increased paired-pulse facilitation indicating a presynaptic change. Records are shown from two separate experiments, in which effects of adenosine and CHA were tested. Note in both cases the increased size of paired-pulse facilitation (size of 2nd response relative to 1st) induced by adenosine or CHA compared with those obtained under control, predrug conditions.

Adenosine inhibition was mediated by PTX-sensitive G-protein.

Because adenosine A1 receptors have been reported to be coupled to pertussis toxin (PTX)-sensitive G-proteins, we tested whether incubation of the tectum with PTX (1 µg/ml) for 2.5-3 h could abolish the inhibition induced by adenosine and CHA. Sample records are shown in Fig. 5, A and B. In PTX-treated tectum, adenosine at 100 µM produced a small, nonsignificant depression (2.35 ± 0.65%, n = 4, P > 0.05) in the amplitude of the field potential, which was significantly smaller than that produced in the untreated opposite tectum (P < 0.05). This result suggests that the adenosine inhibition was mediated by receptors that are coupled to PTX-sensitive G-proteins.


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FIG. 5. Pertussis toxin (PTX) blocks inhibition produced by adenosine and CHA. Sample records show inhibition produced by 100 µM adenosine (A) and 50 nM CHA (B) under control conditions (a) and in preparations pretreated with 1 µg/ml PTX for 2.5-3 h (b). Note that inhibitory effects of adenosine and CHA were largely eliminated by PTX.

Demonstration of inhibition by endogenous adenosine

If there is sufficient endogenous adenosine present in tectum to inhibit synaptic transmission, then the selective adenosine A1 receptor antagonist DPCPX, by itself, should increase the amplitude of the field potential. The results verified the inhibitory effect of endogenous adenosine on synaptic transmission. In these experiments, we stimulated at a slightly higher frequency (1 Hz) to increase the potential release and accumulation of endogenous adenosine. DPCPX at 100 nM, the effective concentration for blocking exogenous adenosine, consistently produced a small, yet significant increase in field potential amplitude (14 ± 3%, n = 5, P < 0.01). An example of the DPCPX effect is shown in Fig. 6A and pooled data in Fig. 6B.


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FIG. 6. Evidence for inhibition by endogenous adenosine. A: selective A1 receptor antagonist DPCPX (100 nM) causes increased field potential amplitude. Field potentials were evoked by supramaximum stimulation at 1 Hz rather than 0.2 Hz to increase potential release of endogenous adenosine. Increased amplitude suggests a tonic inhibition by endogenous adenosine acting on A1 receptors. B: pooled data of 4 such experiments.

Activation of C-kinase attenuated adenosine inhibition

C-kinase phosphorylates a large array of receptors and signal transduction molecules, including the PTX-sensitive G-proteins (Katada et al. 1985). We therefore tested whether or not the inhibitory action of adenosine at the retinotectal synapse is also under the control of C-kinase. Pretreatment of the preparation with C-kinase-activating tetradecanoyl phorbol acetate (TPA; 1 µM) greatly decreased the inhibitory effect of adenosine and CHA (Fig. 7), as reported elsewhere (Barrie and Nicholls 1993; Budd and Nicholls 1995; Sanchez-Prieto et al. 1996). The adenosine inhibition was 5.4 ± 1.0% compared with 21 ± 2.3% in the opposite control tecta (n = 5, P < 0.001). In contrast, TPA had no effect on the augmentation induced by the selective A2 agonist DPMA (data not shown).


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FIG. 7. Activation of protein kinase C (PKC) blocks inhibition by adenosine and CHA. Sample records show that inhibition produced by 100 µM adenosine (Aa) and 50 nM CHA (Ba) is largely eliminated after treatment with 1 µM TPA to activate PKC (Ab and Bb).

In accordance with this finding, we found that in one batch of fish it was necessary to pretreat the tectum with staurosporine to observe the inhibitory action of CHA. In the absence of staurosporine, CHA only produced a small, insignificant depression of the field potential (1.9 ± 1.7%, n = 6). In the presence of staurosporine (100 nM), the inhibition was 19.6 ± 1.9% (n = 11, P < 0.001). Because staurosporine inhibits several protein kinases including C-kinase, this interesting finding provided further evidence for the involvement of a kinase, possibly C-kinase, in the control of the mechanism mediating the inhibitory effect of adenosine. In this batch of fish, C-kinase may have been constantly activated so that the inhibitory action of adenosine receptors could be seen only when the C-kinase was inhibited by staurosporine.

LTP and adenosine inhibition in regenerating projections

Synaptic responses in the regenerating projection are fluctuating and subject to extremely rapid fatigue, but after induction of long-term potentiation (LTP) become stable and resistant to fatigue (Schmidt 1990). Therefore we tested for sensitivity to adenosine presynaptic inhibition and for any possible changes that might occur during LTP induction.

Application of adenosine to regenerating projections (30-46 days postcrush) caused an average 30.0 ± 12.6% decrease in field potential amplitude, very similar to the decrease seen in normals. An example is shown in Fig. 8A1. In the same tecta or the opposite tecta from the same fish, LTP was induced (using trains of 20 supramaximal stimuli at 0.1 Hz) for an average increase of 161 ± 31% in amplitude (n = 4), as seen previously (Schmidt 1990). After LTP, adenosine application did not decrease transmission, but most often increased it (average of +5.8 ± 4.3%, n = 4; Fig. 8A2). Thus LTP consistently uncouples adenosine inhibition.


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FIG. 8. Effect of long-term potentiation (LTP) and phosphatase inhibition on adenosine inhibition in regenerating retinotectal projection. A1: adensosine at 200 µM reversibly inhibits responses of regenerating projection before LTP. A2: after LTP in opposite tectum, same adenosine treatment did not inhibit but actually augments transmission. B1: single responses after 1 h treatment with phosphatase inhibitor okadaic acid at 40 nM. Note successively larger responses with each activation. B2: after response stabilized after okadaic acid treatment, adenosine at 200 µM failed to inhibit and again produced a small augmentation. Responses are averages of 2-5 traces (except for single traces in top right). Conventions as in Fig. 1.

In four additional cases, we treated tecta with 40 nM okadaic acid, an inhibitor of phosphatases 1 and 2A, to promote potentiation. After 1 h of okadaic acid treatment without stimulation, resumption of stimulation showed that field potentials became progressively larger with each succeeding stimulus (Fig. 8B1) and there was no need for trains of stimuli to induce LTP. After this okadaic acid-induced LTP, adenosine again produced no inhibition (Fig. 8B2), but instead produced an augmentation of the synaptic responses (average of +9.9 ± 6.3%, n = 4 cases). Thus potentiation is promoted by increasing phosphorylation (via phosphatase inhibition) and again resulted in uncoupling of adenosine inhibition.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

In the present study, we have demonstrated much of the mechanism whereby adenosine inhibits transmission at the goldfish retinotectal synapse and explored its role in maturation of synapses. Specifically we have shown that 1) adenosine acts via the A1 receptor subtype, 2) the A1 receptors are coupled via PTX-sensitive G protein, 3) transmission is mediated predominantly by N-type Ca2+ channels, known to be modulated by these G-proteins, 4) the adenosine inhibition is primarily presynaptic as judged by paired-pulse facilitation, 5) endogenous adenosine limits transmission even at low frequencies of stimulation, 6) the inhibitory mechanism is uncoupled by activation of C-kinase, and 7) LTP in the regenerating projection removes this adenosine inhibition, greatly altering the properties of the immature synapses. The present study provides support for the hypothesis that adenosine is a major autoinhibitory modulator of synapses and for the first time extends these findings to the visual system. We will first compare the mechanisms found here to those reported in other areas, then discuss the changes in adenosine inhibition seen during LTP induction, and finally relate the inhibitory adenosine modulation and excitatory ACh modulation to the visual processing that takes place in the tectum.

Types of Ca2+ channels mediating retinotectal transmission

Glutamatergic transmission in CNS is usually controlled by a mixture of Ca2+ channel types, such that omega -Ctx GVIA (N-type blocker) alone reduces transmission by 30-70% and omega -Agatx IVA (P-, Q-type blocker) alone decreases by 80-95%. The effects of the two generally do not add linearly, suggesting that two or more channel types are present at many release sites and both are often necessary to trigger release (Dunlap et al. 1995; Reuter 1996; Wheeler et al. 1994). In contrast, the goldfish retinotectal synapse is unusual in its overwhelming reliance on only N-type channels and in its seeming lack of contribution from P or Q types. The nonlinear effect does not allow us to rule out a small contribution from other channel types, but it is likely that P- and Q-type channels are not present. A patch-clamp study (Bindokas and Ishida 1996) showed only T-, L- and N-type Ca2+ channels in goldfish retinal ganglion cell somas, although another nonstandard Ca2+ channel is present in mouse ganglion cells (Rothe and Grantyn 1994).

The goldfish retinotectal synapse also differs, but less so, from that of the frog, which was examined by Feller et al. (1996). There, omega -Ctx GVIA decreased the current by 60-70% and the transmission by 70%, whereas omega -Agatx IVA had only a small effect on current and no effect on transmission. The authors concluded that a nonN-type channel was contributing significantly to transmission in frog. The predominance of the N-type Ca2+ channel in goldfish lends itself to adenosine modulation, because it is commonly preferentially affected by adenosine (Dolphin 1995; Mogul et al. 1993; Toth et al. 1996; Yawo and Chuhma 1993, but see Zong et al. 1995).

Mechanism of presynaptic inhibition

Presynaptic inhibition may occur via different mechanisms that decrease Ca2+ entry: via directly inhibiting Ca2+ channels or via increasing K+ conductance in the presynaptic nerve terminals to limit Ca2+ channel activation. Previous studies have shown that the former was the major pathway for adenosine receptor-mediated presynaptic inhibition (Yawo and Chuhma 1993) and for neurotransmitter or modulator mediated presynaptic inhibition in general (see review by Wu and Saggau 1997).

Protein kinase C (PKC) is involved in the regulation of many biological processes, including presynaptic transmitter release. The present study extends PKC modulation to the A1 adenosine receptor-mediated inhibition in the goldfish retinotectal synapse. Activation of presynaptic class I metabotropic glutamate receptors coupled to phospholipase C could furnish a pathway for activation of PKC in nerve terminals (Sanchez-Prieto et al. 1996). Substrates for PKC phosphorylation may include membrane adenosine receptors, the G-proteins or the ion channels and in the present experiment we were not able to distinguish among these possibilities. However, it seems likely that the inhibitory G-protein is the primary target of PKC phosphorylation. In the cat sympathetic ganglion, PKC activation greatly depresses the inhibition produced by opioid and adrenergic receptors that are G-protein-coupled and PTX-sensitive, but not that produced by gamma -aminobutyric acid (GABAA) receptors, which are not coupled to G-proteins (Zhang et al. 1996). These findings suggest that the target of PKC phosphorylation is most likely downstream from different membrane receptors and acts at some molecule on which the pathways activated by different receptors converge-presumably the G-protein. Indeed, it has been reported that PKC phosphorylates the inhibitory G-protein and thereby interferes with its function (Katada et al. 1985). That possibility is consistent with our findings here of PTX sensitivity of the adenosine inhibition.

Interestingly, studies of mammalian retinotectal projection have demonstrated only adenosine augmentation and not inhibition of retinotectal transmission (Ishikawa et al. 1997). The augmentation was blocked by a selective A2 antagonist. It is not clear why no A1-mediated inhibition was seen because A1 receptors were demonstrated on retinotectal terminals in this species (i.e., rat) (Braas et al. 1987; Wan and Geiger 1990) and because Northern blots verified A1 receptor expression in tectal cells (Ishikawa et al. 1997). In our experiments, A1-mediated inhibition generally overwhelmed the A2-mediated augmentation, except when phorbol treatment was used to uncouple the A1 receptor or in one group of fish where tonic PKC activation apparently eliminated the A1 response, which returned after staurosporine treatment. The predominance of the A1 response could be due to several possible factors: 1) A1 receptors might outnumber A2 receptors, 2) A1 receptors might be more efficiently coupled to the transduction machinery (because of prevalence of G-protein types present), or 3) the A1 receptors, known to be localized to the presynaptic terminal (Goodman et al. 1983; Wan and Geiger 1990), might have a more immediate effect than the A2 receptors, which must be localized to postsynaptic tectal neurons or glia.

Functional demonstration of significant endogenous adenosine

Endogenous adenosine levels are sufficient to inhibit excitatory synaptic transmission in hippocampal slices: adenosine A1 antagonists caused increased excitatory postsynaptic potentials (EPSPs) in the dentate gyrus (Prince and Stevens 1992) and in area CA1 (Dunwiddie and Diao 1994). In the latter case, the increase of 18-20% was similar to the 14% increase seen here. Although there have been no studies on the localization of adenosine to the goldfish retinotectal synapse, both adenosine uptake studies and immunohistochemisty show that endogenous adenosine is present in ganglion cells of several mammalian species (Blazynski et al. 1989; Braas et al. 1987; Goodman et al. 1983) and thus is likely to be present in their axon terminals and possibly released on stimulation. In addition, ATP is sometimes released with neurotransmitters and then converted to adenosine after release. The exact extracellular concentration of adenosine in slices or in tectum here is hard to judge because adenosine is continually being taken up by transporters and broken down by adenosine deaminase in the tissue (Dunwiddie and Diao 1994). Nevertheless, our results are in agreement that levels of adenosine are sufficient to furnish a tonic inhibitory tone, which would presumably fluctuate somewhat with the frequency of impulses in the projection. Here it was significant even at 1 Hz in normal tectum. In immature projections, uptake might be less efficient, so that tonic effects might be greater still.

Adenosine effect in regenerating projections and the effect of LTP

The regenerating retinal axons initially produce widespread branches (Schmidt et al. 1988) that explore a wide area of tectum and then go through an activity-driven sharpening (pruning of branches) during a sensitive period beginning when synaptic transmission is reestablished (Eisele and Schmidt 1988; Schmidt and Buzzard 1990). We have also demonstrated similar effects of manipulating activity in the developing projection (Schmidt and Buzzard 1993). This sensitive period is associated with a remarkable capacity for long-term potentiation (LTP) that may serve as the first step in stabilizing synapses in the retinotopically correct area by reinforcing synapses carrying activity correlated with that of neighboring synapses from neighboring ganglion cells in the retina (Schmidt 1990). This potentiation converts the small, highly fluctuating synaptic responses into larger and much more consistent responses, like those in mature tectum. Because fluctuating presynaptic release is the most likely cause of the fluctuating response and because levels of endogenous adenosine are sufficient to depress transmission even in the mature projection, it is likely that adenosine is even more effective at the immature synapses where the uptake machinery has not yet matured. In addition, we have also found that metabotropic glutamate receptors furnish a parallel autoinhibition of presynaptic release (Schmidt 1995). Elimination of both forms of autoinhibition could therefore account both for the increased consistency of responses and for some of the increase in response amplitude.

Our results show the likely mechanism by which adenosine autoinhibition is eliminated. In normal projections, activation of PKC largely eliminates adenosine inhibition and LTP in this system probably requires PKC activation, because it is prevented by PKC blockers (unpublished data). In fact, we have recently found that during regeneration PKC expression is strongly up-regulated in retinal ganglion cell axons and in normal fish high PKC is also present within the immature, newly added ganglion cell axons coming from the growth zone in peripheral retina (Schmidt 1993 and J. T. Schmidt, unpublished observations). Some lower amount of PKC must also be present in normal mature optic axons because activation of PKC effectively eliminates adenosine inhibition and indeed all ganglion cell somas stain strongly for PKC, although their axons stain poorly (Schmidt 1993 and J. T. Schmidt, unpublished observations).

Further evidence for a phosphorylation mechanism in LTP induction can be found in the effects of inhibiting protein phosphatases 1 and 2A with okadaic acid. (At 40 nM the inhibition was probably largely protein phosphatase 1) This treatment dramatically triggered LTP induction after only a very few test stimuli and is consistent with PKC augmenting Ca2+ influx through Ca2+ channels by removing the G-protein mediated inhibition (Budd and Nicholls 1995; Murakami et al. 1994; Swartz 1993). PKC activation alone via phorbol treatment of tectum in our experiments caused only a small increase in transmission, but okadaic acid was much more effective. This parallels the effects of these agents on Ca2+ channels in hippocampal synaptosomes (Barschat and Rhodes 1995) and suggests that the activity of presynaptic Ca2+ channels are under dynamic bidirectional control but may be influenced more strongly by highly active phosphatases. The okadaic acid-induced potentiation behaved like LTP in that it resulted in a stable and larger responses and eliminated the inhibitory effect of adenosine. In nearly all cases, the adenosine response was converted to an augmentation, which is consistent with an uncovering of the A2 receptor subtype, because A2 agonists produced augmentation even in normal projections. In fact, Sekino et al. (1991) found that A2 receptors were necessary for induction of LTP of the field EPSP of hippocampus. Differential adenosine responses (inhibiting release via A1 receptors at low activity and augmenting release via A2 receptors at higher activity levels or after LTP induction) could help explain activity-dependent competition between synaptic terminals. Terminals in which LTP has removed A1-induced inhibition would have a distinct advantage in competition with other terminals that were inhibited by A1 receptors. The conversion of the adenosine autoinhibition to an augmentation can also account for most of the changes in response characteristics as the inconstant immature responses become more stable and mature, which would also make them more effective at inducing further increases in effectiveness.

Role of adenosine modulation and ACh modulation in visual processing

Goodman et al. (1983) noted that, in the rat, adenosine A1 receptors were localized on the retinal terminals in the mammalian tectum but not on the retinal terminals in the lateral geniculate nucleus, which may have significance for visual processing in the two structures. Although lateral geniculate neurons relay visual information to cortex with receptive fields very similar to those of retinal ganglion cells, neurons in tectum have receptive fields far different from their retinal fiber inputs, featuring prominent habituation and repetitive firing to retinal input (Guthrie and Banks 1974; Niida et al. 1980). This transformation in tectum may result in part because of the dual, bidirectional presynaptic modulation. We previously showed that nicotinic cholinergic receptors on retinal terminals can enhance retinotectal transmission and even cause reactivation of the synapse (King and Schmidt 1991a), and that the enhancement/reactivation is visually driven by feedback loops both through type XIV neurons in deep tectum and through Nucleus Isthmi (King and Schmidt 1991b; King and Schmidt 1993). This bidirectional control may be important in selection of targets for visually elicited eye movements, for visual orienting, and for prey capture, all of which are known to be functions of the tectum.

    ACKNOWLEDGEMENTS

  We thank Drs. Ben Szaro and Suzannah Tieman for useful comments on the manuscript and M. Buzzard for technical assistance.

  This work was supported by National Eye Institute Grant EY-03736 to J. T. Schmidt.

    FOOTNOTES

  Address for reprint requests: J. T. Schmidt, Dept. of Biological Sciences and Neurobiology Research Center, SUNY, 1400 Washington Ave., Albany, NY 12222.

  Received 25 August 1997; accepted in final form 31 October 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society