Presynaptic inhibitory receptors mediate the depression of synaptic transmission upon hypoxia in rat hippocampal slices

Hypoxia markedly depresses synaptic transmission in hippocampal slices of the rat. This depression is attributed to presynaptic inhibition of glutamate release and is largely mediated by adenosine released during hypoxia acting through presynaptic adenosine A(1) receptors. Paired pulse facilitation studies allowed us to confirm the presynaptic nature of the depression of synaptic transmission during hypoxia. We tested the hypothesis that activation of heterosynaptic inhibitory receptors localized in glutamatergic presynaptic terminals in the hippocampus, namely gamma-aminobutyric acid subtype B (GABA(B)) receptors, alpha(2)-adrenergic receptors, and muscarinic receptors might contribute to the hypoxia-induced depression of synaptic transmission. Field excitatory postsynaptic potentials were recorded in the CA1 area of hippocampal slices from young adult (5-6 weeks) Wistar rats. Neither the selective antagonist for alpha(2)-adrenergic receptors, rauwolscine (10 microM), nor the antagonist for the GABA(B) receptors, CGP 55845 (10 microM), modified the response to hypoxia. The selective adenosine A(1) receptor antagonist, DPCPX (50 nM), reduced the hypoxia-induced depression of synaptic transmission to 59.2+/-9.6%, and the muscarinic receptor antagonist, atropine (10 microM), in the presence of DPCPX (50 nM), further attenuated the depression of synaptic transmission to 49.4+/-8.0%. In the same experimental conditions, in the presence of DPCPX (50 nM), the muscarinic M(2) receptor antagonist AF-DX 116 (10 microM), but not the M(1) receptor antagonist pirenzepine (1 microM), also attenuated the hypoxia-induced depression to 41.6+/-6.6%. Activation of muscarinic M(2) receptors contributes to the depression of synaptic transmission upon hypoxia. This effect should assume particular relevance during prolonged periods of hypoxia when other mechanisms may become less efficient.     

Changes in hippocampal adenosine efflux, ATP levels, and synaptic transmission induced by increased temperature

Previous studies have demonstrated that when the temperature of hippocampal brain slices is increased, there is a corresponding depression of synaptic potentials mediated by an increased activation of presynaptic adenosine A(1) receptors. The present experiments demonstrate that when the temperature of hippocampal slices is raised from 32.5 degrees C to either 38.5 degrees C or 40.0 degrees C there is a marked, temperature-dependent increase in the efflux of endogenous adenosine and a corresponding decrease in excitatory synaptic responses. The increase in efflux is rapidly reversible on lowering the slice temperature and the temperature-induced efflux is repeatable. Control experiments suggest that this increased efflux of adenosine is not the result of hypoxia or ischemia secondary to a temperature-induced increase in the metabolic rate of the slice. The increase in adenosine efflux was not accompanied by any significant change in the ATP levels in the brain slice, whereas a hypoxic stimulus sufficient to produce a comparable depression of excitatory transmission produced an approximately 75% decrease in ATP levels. These experiments indicate that changes in brain slice temperature can alter purine metabolism in such a way as to increase the adenosine concentration in the extracellular space, as well as adenosine efflux from hippocampal slices, in the absence of significant changes in ATP levels.     

Adrenoceptor subtype-specific acceleration of the hypoxic depression of excitatory synaptic transmission in area CA1 of the rat hippocampus

The depression of excitatory synaptic transmission by hypoxia in area CA1 of the hippocampus is largely dependent upon the activation of adenosine A(1) receptors on presynaptic glutamatergic terminals. As well as adenosine, norepinephrine levels increase in the hypoxic/ischemic hippocampus. We sought to determine the influence of alpha- and beta-adrenoceptor (AR) activation on the hypoxic depression of synaptic transmission utilizing electrophysiological, pharmacological and adenosine sensor techniques. Norepinephrine depressed synaptic transmission and significantly accelerated the hypoxic depression of synaptic transmission. The alpha-AR agonist 6-fluoronorepinephrine mimicked both of these effects whilst the alpha(2)-AR antagonist yohimbine, but not the alpha(1)-AR antagonist urapidil, prevented the actions of 6-fluoronorepinephrine. In contrast, the beta-AR agonist isoproterenol enhanced synaptic transmission and only accelerated the hypoxic depression of transmission in hypoxia-conditioned slices in which the hypoxic release of adenosine is reduced. The effects of isoproterenol were blocked by the non-selective beta-AR antagonist propranolol and the selective beta(1)-AR antagonist betaxolol. Using an enzyme-based adenosine sensor we observed that the application of the beta-AR agonist resulted in increased extracellular adenosine during repeated hypoxia. Our results suggest that alpha(2)-AR activation facilitates the hypoxic depression of synaptic transmission probably via the known alpha(2)-AR-mediated inhibition of presynaptic calcium channels whereas beta(1)-AR activation does so via increased extracellular adenosine and greater activation of inhibitory adenosine A(1) receptors.     

Suppression of presynaptic calcium currents by hypoxia in hippocampal tissue slices.

We tested the hypothesis that suppression of inward calcium current in presynaptic terminals is the cause of failure of synaptic transmission early during cerebral hypoxia. Postsynaptic responses in CA1 zone of hippocampal tissue slices were blocked either by the combined administration of 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 3-((+-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) or by lowering extracellular calcium concentration ([Ca2+]o). Repetitive orthodromic activation of central neurons caused transient decrease of [Ca2+]o (measured by ion selective microelectrodes) in neuropil, attributable to influx of Ca2+ in presynaptic terminals. Presynaptic [Ca2+]o responses were rapidly and reversibly suppressed when oxygen was withdrawn from hippocampal tissue slices. The 'resting' baseline level of [Ca2+]o declined at first gradually, then precipitously as in spreading depression (SD). Presynaptic volleys during high frequency train stimulation were also depressed somewhat before SD began. We conclude that (1) presynaptic Ca2+ currents fail during hypoxia, perhaps because 'resting' intracellular free Ca2+ activity is increased and, in part, also because of partial failure of presynaptic impulse conduction; (2) the influx of Ca2+ into brain cells in hypoxic spreading depression is not mediated by glutamate/aspartate dependent channels.     

Adenosine by activating A1 receptors prevents GABAA-mediated actions during hypoxia in the rat hippocampus

The relative contribution of adenosine and γ-aminobutyric acid (GABA) for the hypoxia-induced depression of field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices, was investigated. It is concluded that both adenosine and GABA, by activating A₁ and GABA_A receptors, could be responsible for the inhibition of synaptic transmission during hypoxia, but the action of endogenous GABA becomes evident only when the adenosine A₁ receptor action is precluded.     

Doc2alpha is an activity-dependent modulator of excitatory synaptic transmission

Doc2alpha is a synaptic vesicle-associated Ca2 + -binding protein. To study the role of Doc2alpha in synaptic transmission and modulation, we generated homozygous null Doc2alpha mutant mice. In the CA1 region of hippocampal slices in the mutant mice, excitatory synaptic responses evoked with prolonged 5 Hz stimulation showed a significantly larger frequency facilitation followed by a steeper depression than those in wild-type mice, whereas there was no difference in synaptic transmission at lower frequencies or in paired-pulse facilitation. These results suggest that Doc2alpha regulates synaptic transmission when high Ca2 + concentrations in the presynaptic terminal are sustained. Furthermore, the mutant mice showed impairment in long-term potentiation and passive avoidance task. Thus, Doc2alpha may regulate transmitter release during repetitive synaptic activation, thereby contributing to memory formation.     

Differential effect of adenosine on pre- and postsynaptic calcium fluxes

In rat hippocampal slices, stimulus-evoked field potentials and the concomitant decrease of the extracellular concentration of free Ca ions [Ca2+]o were measured with combined reference/ion-sensitive microelectrodes. By reducing [Ca2+]o from 2.0 mM to 0.2 mM, evoked synaptic transmission was blocked, but orthodromic repetitive stimulation of CA1 afferents still elicited a marked decrease of [Ca2+]o. This Ca2+ signal is attributed predominantly to Ca2+ entry into the activated axon terminals. It was significantly depressed by adenosine. The adenosine agonist, L-phenylisopropyl adenosine (L-PIA) was more effective than D-PIA, indicating that the adenosine depression of presynaptic Ca2+ entry is mediated via the A1 receptor. 4-Aminopyridine (4-AP) enhanced decreases in [Ca2+]o without restoring synaptic transmission. Adenosine depressed also these Ca2+ signals. Adenosine deaminase was even more effective in the presence of 4-AP and enhanced the orthodromic Ca2+-signal by a factor of two. Antidromic stimulation of hippocampal pyramidal cells also evoked reductions in [Ca2+]o. These were less affected by adenosine and the other treatments under the conditions tested.     

Stabilizing effects of extracellular ATP on synaptic efficacy and plasticity in hippocampal pyramidal neurons

The role of adenosine triphosphate (ATP) as a neurotransmitter and extracellular diffusible messenger has recently received considerable attention because of its possible participation in the regulation of synaptic plasticity. However, the possible contribution of extracellular ATP in maintaining and regulating synaptic efficacy during intracellular ATP depletion is understudied. We tested the effects of extracellular ATP on excitatory postsynaptic currents (EPSCs) evoked in CA1 pyramidal neurons by Schaffer collateral stimulation. In the absence of intracellular ATP, EPSC rundown was neutralized when a low concentration of ATP (1 microm) was added to the extracellular solution. Adenosine and ATP analogues did not prevent the EPSC rundown. The P(2) antagonists piridoxal-5'-phosphate-azophenyl 2',4'-disulphonate (PPADS) and reactive blue-2, and the P(1) adenosine receptor antagonist 8-cyclopentyltheophylline (CPT) had no detectable effects in cells depleted of ATP. However, the protective action of extracellular ATP on synaptic efficacy was blocked by extracellular application of the protein kinase inhibitors K252b and staurosporine. In contrast, K252b and staurosporine per se did not interfere with synaptic transmission in ATP loaded cells. Without intracellular ATP, bath-applied caffeine induced a transient (< 35 min) EPSC potentiation that was transformed into a persistent long-term potentiation (> 80 min) when 1 microm ATP was added extracellularly. An increased probability of transmitter release paralleled the long-term potentiation induced by caffeine, suggesting that it originated presynaptically. Therefore, we conclude that extracellular ATP may operate to maintain and regulate synaptic efficacy and plasticity in conditions of abnormal intracellular ATP depletion by phosphorylation of a surface protein substrate via activation of ecto-protein kinases.     

New roles for astrocytes: regulation of synaptic transmission

Abstract Although glia often envelop synapses, they have traditionally been viewed as passive participants in synaptic function. Recent evidence has demonstrated, however, that there is a dynamic two-way communication between glia and neurons at the synapse. Neurotransmitters released from presynaptic neurons evoke Ca2+ concentration increases in adjacent glia. Activated glia, in turn, release transmitters, including glutamate and ATP. These gliotransmitters feed back onto the presynaptic terminal either to enhance or to depress further release of neurotransmitter. Transmitters released from glia can also directly stimulate postsynaptic neurons, producing either excitatory or inhibitory responses. Based on these new findings, glia should be considered an active partner at the synapse, dynamically regulating synaptic transmission.     

Imaging of synaptically evoked intrinsic optical signals in hippocampal slices.

Imaging analysis techniques were used to examine changes in the intrinsic optical properties in hippocampal brain slices that occurred during synaptic activity evoked by Schaffer collateral stimulation in CA1. Repetitive synaptic activity was associated with an increase in light transmission in the synaptic region in stratum radiatum. The effect was seen at wavelengths of light between 450 and 800 nm but was of greater amplitude at longer wavelengths. Blocking synaptic transmission with either Ca(2+)-free EGTA perfusate or kynurenic acid (an excitatory amino acid antagonist) blocked the optical signal, indicating that it resulted from postsynaptic activation of the cells and was not due to presynaptic fiber volleys or transmitter release alone. Because the optical changes were blocked by reducing extracellular Cl- (by replacement with gluconate) or by furosemide (an anion transport inhibitor), increased Cl- transport (conceivably Na-K-2Cl cotransport) may generate these signals possibly by causing cellular swelling and thereby less light scattering. These optical changes were not blocked, however, by bicarbonate-free solution, indicating that bicarbonate transport may not be involved. Changes in the intrinsic optical signal could be related to glial swelling due to K+ released during neuronal activity because high-K(+)-induced swelling of cultured astrocytes is blocked by furosemide and low-Cl- solution. Intrinsic optical signals of neuronal tissue should be considered when voltage- or ion-sensitive dyes are used.     

The adenosine antagonist 8-cyclopentyltheophylline reduces the depression of hippocampal neuronal responses during hypoxia

Exposure of rat hippocampal slices to hypoxic conditions for 15 min produced a rapid, but completely reversible depression of evoked synaptic potentials. The specific A₁ adenosine receptor antagonist 8-cyclopentyltheophyline (8-CPT) significantly reduced hypoxia-induced synaptic depression in concentration-dependent manner. It is concluded that adenosine, which is neuroprotective when exogenously applied during severe hypoxia because of its ability to depress synaptic transmission, may have an important and exploitable endogenous role in the protection of sensitive neurons.     

Neuropeptide Y action in the rat hippocampal slice: site and mechanism of presynaptic inhibition

Neuropeptide Y (NPY), the most abundant peptide in mammalian CNS, has been shown to inhibit excitatory neurotransmission presynaptically at the stratum radiatum-CA1 synapse in the in vitro rat hippocampal slice. We examined the site and mechanism of this inhibition in a series of in vitro intra- and extracellular recordings in areas CA1 and CA3, the source of much of the excitatory synaptic input to the CA1 neurons. NPY's inhibitory action at the stratum radiatum-CA1 synapse was unaffected by high concentrations of the antagonists bicuculline, theophylline, or atropine, suggesting that it does not act by stimulating the release of the known presynaptic inhibitory transmitters GABA, adenosine, or ACh, respectively. Bath application of 10(-6) NPY, a concentration that strongly inhibited the stratum radiatum-CA1 synapse had no effect on CA3 neuron resting potential, input resistance or action potential amplitude, threshold, or duration. NPY also does not alter the amplitude or duration of the prolonged CA3 action potentials evoked in the presence of TTX, tetraethyl-ammonium, and elevated external Ca2+ or those evoked in the presence of TTX and Ba2+ ions. NPY therefore does not alter the passive or active properties of the somata of the presynaptic CA3 neurons. Neither the afferent fiber volley of the Schaffer collaterals in stratum radiatum of area CA1 nor the excitability of the CA3 terminals in CA1 was affected by NPY application. However, application of the transient K+ current blocker, 4-aminopyridine (4-AP) at concentrations of 10 and 50 microM, completely abolished the action of 10(-6) M NPY on the stratum radiatum-CA1 excitatory synaptic potentials. This action of 4-AP could be reversed by reducing extracellular Ca2+ concentrations from a control level of 1.5 to 0.7 mM (in 10 microM 4-AP) and to 0.5 mM (in 50 microM 4-AP). The evidence suggests that NPY inhibits excitatory synaptic transmission at the Schaffer collateral-CA1 synapse by acting directly at the terminal to reduce a Ca2+ influx.     

Stimulation-dependent release of adenosine triphosphate from hippocampal slices

Schaffer collaterals of rat and mouse hippocampal slices were stimulated with bursts of pulses (300 Hz for 50 ms, 2-s intervals) for 30-s which caused a stable increase in the size of the population spike known as long-term potentiation. The release of adenosine triphosphate (ATP) was measured with a luciferase-luciferine system and the light emitted was recorded with a photomultiplier placed beneath a modified slice chamber. ATP release was observed shortly after the start of stimulation and was quantified by comparison with the response of standard solutions of ATP. No ATP release was observed in a Ca2+ free solution or after low frequency stimulation (1 Hz). Glutamate (2 mM), applied without electrical stimulation, did not evoke ATP release. Also, the glutamate receptor blocker, kynurenic acid (10 mM), did not block ATP release. It is concluded that ATP is released from electrically stimulated hippocampal slices from presynaptic nerve terminals in a calcium-dependent fashion and may play a role in the modulation of synaptic efficiency.     

Basal levels of adenosine modulate mGluR5 on rat hippocampal astrocytes

Neuronal activity elicits increases in intracellular Ca2+ in astrocytes, which in turn can elevate neuronal Ca2+ and potentiate the efficacy of excitatory synaptic transmission. Therefore, understanding the modulation of astrocyte Ca2+ elevations by neurotransmitters should aid in understanding astrocyte-neuronal interactions. On cultured hippocampal microislands containing only astrocytes, activation of metabotropic glutamate receptors (mGluRs) with the specific agonist 1S,3R-ACPD triggers Ca2+ elevations that are potentiated by adenosine A1 receptor activation. A1 receptor modulation of mGluR-induced Ca2+ elevations is blocked by pertussis toxin and is mimicked by the wasp venom peptide mastoparan, suggesting that potentiation occurs by means of a G(i/o) mechanism. Surprisingly, on microislands containing only astrocytes, A1 receptor antagonism or adenosine degradation suppresses mGluR-triggered Ca2+ elevations, strongly suggesting that astrocytes are a source of physiologically relevant concentrations of adenosine.     

Phorbol ester alters the electrophysiological responses to hypoxia and ischemic-like conditions in the rat hippocampal slice

The effect of incubation with the protein kinase C activator, 4β-phorbol 12,13-dibutyrate (β-PDBu) on the electrophysiological responses to hypoxia and combined hypoxia and hypoglycemia was investigated in the rat hippocampal slice. Preincubation with β-PDBu prevents adenosine-mediated inhibition of synaptic transmission under normoxic, normoglycemic conditions. β-PDBu preincubation also reduces the adenosine-mediated hypoxia-induced depression of synaptic transmission revealing a substantial adenosine-independent hypoxia-induced depression of synaptic transmission. During combined hypoxia and hypoglycemia, slices preincubated in β-PDBu display a significant shortening of the time to anoxic depolarization, an effect of β-PDBu that is not mimicked by application of the adenosine antagonist cyclopentyltheophylline (8-CPT). It is concluded that the state of PKC activation may influence the electrophysiological responses to hypoxia and ischemia.     

Stressor-related impairment of synaptic transmission in hippocampal slices from alpha-synuclein knockout mice

The role of alpha-synuclein (alpha-Syn) has recently received considerable attention because it seems to play a role in Parkinson's disease (PD). Missense mutations in the alpha-Syn gene were found in autosomal dominant PD and alpha-Syn was shown to be a major constituent of protein aggregates in sporadic PD and other synucleinopathies. Under normal conditions, alpha-Syn protein is found exclusively in synaptic terminals. However, the potential participation of alpha-synuclein in maintaining and regulating synaptic efficacy is unknown. We have investigated the excitatory synaptic modulation of alpha-synuclein in CA1 pyramidal neurons, using the in vitro hippocampal slice technique. The 4-aminopyridine-induced increase of both spontaneous excitatory postsynaptic current (EPSC) frequency and amplitude was significantly higher in alpha-Syn wild-type than knockout mice, whereas basal spontaneous EPSC frequency and amplitude was similar in both animals. As the spontaneous synaptic activity was abolished by tetrodotoxin, which indicates that it was a result of action potential-mediated transmitter release from presynaptic terminals, spontaneous EPSC changes observed in alpha-Syn knockout mice suggest that these animals present a modification of synaptic transmission with a presynaptic origin. Presynaptic depression of evoked EPSCs by hypoxia or adenosine was significantly larger in alpha-Syn knockout than in wild-type mice, further supporting the hypothesis of regulation of synaptic transmission by alpha-Syn. Together, these observations indicate that the loss of alpha-Syn reduces synaptic efficacy when the probability of transmitter release is modified. We conclude that alpha-Syn might have important actions on the maintenance of the functional integrity of synaptic transmission and its regulation in hippocampus.     

Chlorpromazine protects brain tissue in hypoxia by delaying spreading depression-mediated calcium influx

We have investigated the possible protective effect of chlorpromazine in hypoxia of brain tissue, using rat hippocampal slices maintained at 35-36 degrees C. The recovery of synaptic transmission along the Schaffer collaterals to the CA1 pathway after 9 min hypoxia was compared in chlorpromazine-treated and in control slices. Recovery upon reoxygenation was the exception in control slices, while it was observed in approximately 50 and 100% of slices treated with 7 and 70 microM chlorpromazine, respectively. Chlorpromazine also significantly delayed the occurrence of the hypoxia-induced spreading depression (SD). Recovery took place when SD occurred late during hypoxia, not when it occurred early. In those slices in which 7 microM chlorpromazine afforded no protection, SD occurred as early as it did in control slices. In further experiments, we deliberately induced SD during hypoxia in 70 microM-treated slices by topically applying a drop of high-K+ artificial cerebrospinal fluid (ACSF). Recovery was not observed when SD was induced early, but it was observed when it was induced near the end of the hypoxic period. Slices exposed to the same period of hypoxia in Ca2+-free ACSF recovered synaptic transmission (even without chlorpromazine treatment) despite early induction of SD. We conclude that: chlorpromazine protects brain tissue from hypoxia-induced irreversible loss of synaptic transmission; it does so by delaying the occurrence of SD, and hence shortening the time spent in the SD-induced depolarized state; and the harm done by SD in hypoxia is related to the influx of Ca2+ into neurons.     

Microglia‐derived purines modulate mossy fibre synaptic transmission and plasticity through P2X4 and A1 receptors

Recent data have provided evidence that microglia, the brain‐resident macrophage‐like cells, modulate neuronal activity in both physiological and pathophysiological conditions, and microglia are therefore now recognized as synaptic partners. Among different neuromodulators, purines, which are produced and released by microglia, have emerged as promising candidates to mediate interactions between microglia and synapses. The cellular effects of purines are mediated through a large family of receptors for adenosine and for ATP (P2 receptors). These receptors are present at brain synapses, but it is unknown whether they can respond to microglia‐derived purines to modulate synaptic transmission and plasticity. Here, we used a simple model of adding immune‐challenged microglia to mouse hippocampal slices to investigate their impact on synaptic transmission and plasticity at hippocampal mossy fibre (MF) synapses onto CA3 pyramidal neurons. MF–CA3 synapses show prominent forms of presynaptic plasticity that are involved in the encoding and retrieval of memory. We demonstrate that microglia‐derived ATP differentially modulates synaptic transmission and short‐term plasticity at MF–CA3 synapses by acting, respectively, on presynaptic P2X₄ receptors and on adenosine A₁ receptors after conversion of extracellular ATP to adenosine. We also report that P2X₄ receptors are densely located in the mossy fibre tract in the dentate gyrus–CA3 circuitry. In conclusion, this study reveals an interplay between microglia‐derived purines and MF–CA3 synapses, and highlights microglia as potent modulators of presynaptic plasticity.     

Temperature-dependent modulation of excitatory transmission in hippocampal slices is mediated by extracellular adenosine.

Although extracellular adenosine concentrations in brain are increased markedly by a variety of stimuli such as hypoxia and ischemia, it has been difficult to demonstrate large increases in adenosine with stimuli that do not result in pathological tissue damage. The present studies demonstrate that increasing the temperature at which rat hippocampal brain slices are maintained (typically from 32.5 to 38.5 degrees C) markedly inhibits excitatory synaptic transmission. This effect was reversible on cooling, readily repeatable, and was blocked by A1 receptor antagonists and by adenosine deaminase, suggesting that it was mediated by increased activation of presynaptic adenosine A1 receptors by endogenous adenosine. This increase in adenosinergic inhibition was not a response to hyperthermia per se, because it could be elicited by temperatures that remained entirely within the hypothermic range (e. g., from 32.5 to 35.5 degrees C). The increased activity at A1 receptors appeared to be attributable to the direct release of adenosine via nucleoside transporters; the release of adenine nucleotides, linked to either the activation of NMDA receptors or the increased efflux of cAMP, appeared not to be involved. These results suggest that changes in brain temperature can alter the regulation of extracellular adenosine in rat brain slices and that increased adenosine release may be an important regulatory mechanism for countering increased excitability consequent to increased brain temperature.     

Late developmental changes in the ability of adenosine A1 receptors to regulate synaptic transmission in the hippocampus

Paired-pulse facilitation (PPF) of CA3–CA1 excitatory postsynaptic potentials (EPSP) was compared in hippocampal slices from juvenile (postnatal day (P) 15–21) and young adult rats (P28–P35) following application of adenosine. Relative to juveniles, young adults expressed an increase in baseline synaptic strength that was accompanied by a decrease in PPF suggesting a developmental increase in transmitter release. While adenosine depressed the EPSP slope to a similar extent in juveniles and young adults, PPF increased during adenosine application only for young adults. The differential effect of adenosine on PPF was not due to differences in receptor function or in extracellular ligand levels, since the A₁ antagonist cyclopentyltheophylline (CPT) did not differentially affect PPF across age. Adenosine could increase PPF in juvenile slices under conditions of enhanced transmitter release, through an increase in the bath Ca²⁺ concentration, or addition of forskolin to the bath. These data indicate that the ability to modify synaptic transmission through presynaptic adenosine A₁ receptors increases across postnatal development with the maturation of release mechanisms.