A cautionary note: the actions of adenosine agonists and antagonists may be reversed under certain conditions in primary cultures

It is now generally accepted that adenosine has a neuroprotective role in the central nervous system. Agonists of adenosine such as 2-chloroadenosine (2-ClA) have been shown to be neuroprotective, while antagonists such as 8-phenyltheophylline (8-PT) increase neurotoxicity. However, paradoxical results have been reported with adenosine analogues, especially with respect to length of time of administration. We observe similarly contradictory findings with respect to 2-ClA and 8-PT actions in primary hippocampal cultures exposed to glutamate or kainic acid. We found 8-PT and 2-ClA had antagonist and agonist actions, respectively, only with acute (1 h) treatment; with chronic treatment (24 h), 2-ClA had no effects, while 8-PT had significant agonist actions. We also show that with variations in the type of culturing system, concentration, and pH that 8-PT's neurotoxic antagonist actions could be dramatically changed. We, therefore, present this paper as a cautionary note in experimenting with adenosine analogues.     

Electrical activity of the reticular formation during aversive and appetitive conditioning in rats

An investigation has been made of the effects of noradrenaline on excitatory transmission at the lateral olfactory tract (LOT)-superficial pyramidal cell synapse of the rat olfactory cortex slice by measuring the effects of bath-applied noradrenaline on the amplitudes and latencies of the field potentials evoked on LOT stimulation. Low concentrations of noradrenaline (0.1-5 microM) facilitate transmission whereas higher doses (20-250 microM) depress transmission. Both these effects were completely blocked by non-selective alpha- and beta-adrenoceptor antagonists, by 2-amino-5-phosphonovaleric acid (an antagonist of excitatory amino acid receptors of the N-methyl-D-aspartate type) and by the methylxanthine theophylline. The depressant effects of noradrenaline were mimicked by bath application of GABA or adenosine and specifically antagonized by bicuculline and picrotoxin. In parallel experiments, noradrenaline (100 microM) significantly increased the potassium-evoked release of endogenous aspartate, glutamate and GABA, proposed transmitters of the olfactory cortex, although the effect on GABA release was specifically antagonized by 2-amino-5-phosphonovaleric acid. Noradrenaline (100 microM) also significantly increased the potassium-evoked release of D-[3H]aspartate, an effect antagonized by a number of alpha- and beta-adrenoceptor antagonists. It is concluded that at low concentrations, noradrenaline facilitates transmission at the LOT-superficial pyramidal cell synapse by increasing excitatory amino acid neurotransmitter release. This effect is mediated by both alpha- and beta-adrenoceptors although the primary site of release is unknown. At higher concentrations of noradrenaline, the increased levels of excitatory transmitters release sufficient endogenous GABA (and possibly adenosine) to cause an overall depression of transmission. These conclusions are supported by the results of a series of experiments in which the effects of noradrenaline on stimulus input-evoked field potential output relationships were assessed. It is not possible to exclude additional direct effects of noradrenaline on membrane excitability.     

GABAB receptor-mediated inhibitory postsynaptic potentials evoked by electrical stimulation and by glutamate stimulation of interneurons in stratum lacunosum-moleculare in hippocampal CA1 pyramidal cells in vitro.

Following micropressure application of glutamate (500 microM) in stratum lacunosum-moleculare (L-M), inhibitory postsynaptic potentials (glut-IPSPs) were recorded in CA1 pyramidal cells. These glut-IPSPs were blocked by tetrodotoxin (1 microM) and, thus, were probably generated by the activation of local interneurons. The effects of pharmacological antagonists on glut-IPSPs and on electrically-evoked early and late IPSPs were assessed in the same cells during the same application of the antagonist. Local application of the GABAB antagonist 2-OH saclofen (1-4 mM) reduced both glut-IPSPs and late IPSPs but not early IPSPs. In contrast, the GABAB antagonist phaclofen (20 mM) reduced late IPSPs but not early IPSPs but not early IPSPs or glut-IPSPs. Early IPSPs were blocked by the GABAA antagonists bicuculline and picrotoxin but late IPSPs and glut-IPSPs were not. Repetitive electrical stimulation depressed early and late IPSPs as well as glut-IPSPs, suggesting that interneurons activated with glutamate were also stimulated electrically. Thus, interneurons in str. lacunosum-moleculare appear to inhibit pyramidal cells via a GABAB receptor-mediated IPSP. The discrepancy in the pharmacological profile of the GABAB glut-IPSPs and of the GABAB late IPSPs may suggest the presence of two GABAB mechanisms in CA1 pyramidal cells.     

Adenosine-amino acid interactions in the chick brain: a role in passive avoidance learning

The present work describes interactions between adenosine and the amino acids glutamate and GABA in slices of intermediate medial hyperstriatum ventrale (IMHV), an area of the chick brain known to be involved in learning and memory events associated with a one-trial passive avoidance task. In slices derived from the IMHV of untrained chicks, the A(1) receptor agonist N(6)-cyclohexyladenosine (CHA; 10 microM) specifically inhibited glutamate release. Conversely, cyclopentyltheophylline (CPT; 100 microM an A(1) antagonist) increased glutamate release from the slices and blocked the CHA-induced inhibition of glutamate. The A(2) receptor agonist 2-p-(2-carboxylethyl)-phenylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) selectively increased glutamate release when applied at 5 microM while it selectively inhibited GABA release at a lower concentration (10 nM). The addition of NMDA to the medium, resulted in increased adenosine release equivalent to that found following stimulation with 50 mM KCl. Both the NMDA and the KCl-induced increases were eliminated by addition of D-2-amino-5 phosphopentanoic acid (D-AP5), an NMDA-receptor antagonist. Slices prepared from the IMHV of chicks following successful training on the task showed enhanced adenosine release 30 min, 1, 3 and 6.5 h after training compared to chicks trained to peck a water-coated bead. The results show that changes in adenosine release from the IMHV accompany memory formation in the chick. We suggest that adenosine-amino acid transmitter interactions potentially via the activation of NMDA receptors, a necessary step in long-term memory formation for the task, may modulate the formation of memory for the one-trial passive avoidance task.     

In vitro evidence that 5-hydroxytryptamine increases efflux of glial glutamate via 5-HT(2A) receptor activation

Recent studies have established the presence of 5-hydroxytryptamine (5-HT)(2A) receptors on glial cells in culture and in the brain in situ. Here we used cultured C6 glioma cells to investigate the possibility that 5-HT(2A) receptors on glia regulate glutamate release from the cell. The efflux of endogenous glutamate from cultured C6 glioma cells was increased by addition of 5-HT in a concentration-dependent manner (maximal effect +200%). The efflux of serine and aspartate was not altered. The effect of 5-HT was mimicked by both the nonselective 5-HT receptor agonist quipazine and the selective 5-HT(2) receptor agonist 4-iodo-2,5-dimethoxyamphetamine (DOI; both 0.01-100 microM). The 5-HT(2A) receptor antagonists ketanserin (1 microM) and spiperone (1 microM) inhibited the glutamate response to 5-HT, quipazine, and DOI, whereas the effect of 5-HT was not inhibited by the 5-HT(2B/C) receptor antagonist SB200646 (1 microM). The effect of 5-HT on glutamate was specific in that it was reduced in low-calcium medium but was not prevented by furosemide (5 mM), which prevents cell swelling-induced glutamate release. Finally, the glutamate uptake inhibitor 2,4,trans-pyrollidine dicarboxylic acid (50 microM) did not block the 5-HT-induced efflux of glutamate, making involvement of glutamate transport unlikely. In conclusion, 5-HT stimulates the efflux of glutamate from C6 glioma cells following 5-HT(2A) receptor activation and involves a calcium-dependent mechanism.     

Neuronal origins of K(+)-evoked amino acid release from cerebellar cultures.

Neuronal cultures from rat cerebellum consisting of approximately 90% glutamatergic granule neurons, 5-7% GABAergic inhibitory interneurons, and 3-5% glial cells, were treated for four days with 50 microM kainic acid (KA) to determine the cellular origin of released endogenous neuroactive substances. KA, known to be selectively toxic to GABAergic neurons, caused an estimated 80% decrease in glutamic acid decarboxylase (GAD) immunofluorescence. Furthermore, K(+)-stimulated release of GABA decreased to 20% of control values, and did not return to control levels in cultures "recovered" two days in KA-free media, suggesting the loss of inhibitory interneurons. Similarly, adenosine and taurine showed decreased K(+)-stimulated release, which was unrecoverable when KA was removed from the medium. K(+)-stimulated release of glutamate and aspartate also decreased by 50% and 70%, respectively, after chronic KA treatment. In contrast, however, this release returned to control levels in recovered cultures. All decreases in K(+)-stimulated release were prevented by concurrent treatment with KA and the KA antagonist 6-cyano-6-nitroquinoxaline-2,3-dione (CNQX), indicating that a receptor-mediated mechanism was involved. We conclude that, in these cultures, most of the K(+)-stimulated release of adenosine and taurine originates from the GABAergic interneurons, the basket and stellate cells, which are selectively killed by the KA treatment. The data also strongly suggest that glutamate and aspartate, the levels of which recover after KA treatment, originate mainly from the granule neurons.     

Dopamine inhibits the release of immunoreactive beta-endorphin from rat hypothalamus in vitro

Mediobasal hypothalamus tissue (MBH) from adult male rats was incubated in Krebs-Ringer bicarbonate medium (KRB). KRB was changed at 15 min intervals and the concentration of immunoreactive beta-endorphin (beta-ENDi) in the medium was measured by radioimmunoassay. Incubation of MBH tissue in normal KRB resulted in a constant release rate of beta-ENDi of approximately 1% of the tissue content per h. KRB containing 45 mM K+ causes a two fold increase in the release rate of beta-ENDi which was Ca2+ dependent. Dopamine (0.01-1.0 microM) inhibits both the spontaneous and the K+-stimulated release of beta-ENDi in a dose related manner. The dopamine receptor blocking agent haloperidol prevents this inhibitory effect of dopamine. The selective D-1 receptor agonist SKF 38393 does not affect the release rate of beta-ENDi; whereas the selective D-2 receptor agonist LY 141865 inhibits both the spontaneous and K+-stimulated release of beta-ENDi. The effects of LY 141865 can be blocked by (-)-sulpiride, a selective D-2 receptor antagonist. Norepinephrine only weakly inhibits the K+-stimulated release of beta-ENDi, an effect that can be blocked by haloperidol but not by the alpha-adrenoceptor blocker phentolamine. At concentrations tested (0.01-1.0 microM), isoproterenol, 5-hydroxytryptamine, carbachol and 8-Br-cAMP (1.0 microM) do not affect beta-ENDi release. It is concluded that dopamine can inhibit the release of beta-ENDi from hypothalamic neurons via a D-2 receptor mechanism.     

The mGlu5 receptor agonist CHPG stimulates striatal glutamate release: possible involvement of A2A receptors

The intrastriatal perfusion of the selective metabotropic glutamate (mGlu)5 receptor agonist (RS)-2-chloro-5-hydroxy-phenylglycine (CHPG, 1000 microM) significantly increased (approximately + 100%, p < 0.05) glutamate extracellular levels with respect to basal values. The potentiating effect of CHPG was prevented by the selective mGlu5 receptor antagonist 2-methyl-6(phenyl-ethynyl)-pyridine (MPEP, 250 microM)) and by the adenosine A2A receptor antagonist SCH 58261 (2 mg/kg, i.p.). The results show that mGlu5 receptors are involved in the regulation of striatal glutamate release and suggest an involvement of adenosine A2A receptors in mGlu5 receptor-mediated effects.     

Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: pharmacological characterization

L-Glutamate neurotoxicity at the N-methyl-D-aspartate (NMDA) receptor was characterized in cultured cerebellar granule cells. When deprived of glucose for 40 min, these cells were killed by 20-60 microM L-glutamate. However, the neurons were resistant to glutamate at concentrations as high as 5 mM when glucose and Mg2+ were present throughout. Both competitive and non-competitive NMDA receptor antagonists completely blocked neurotoxicity due to glutamate and other NMDA receptor agonists. CPP [+/-)-3-(2-carboxypiperazin-4-yl)-prophyl-1-phosphonic acid) was the most effective competitive antagonist with full protection at 100 microM while MK-801 [+/-)-10,11-dihydro-5-methyl-5H-dibenzo[a,d]-cyclohepten-5,10-imin e) was the most effective non-competitive antagonist with full protection at 20 nM. Other antagonists with higher selectivity for other subtypes of glutamate receptors were ineffective. We conclude that glutamate toxicity in energy-deprived cerebellar granule cells is mediated by NMDA receptors. Results are discussed in terms of an hypothesis offering an explanation for the transition of glutamate from neurotransmitter to neurotoxin which emphasizes the responsiveness of the receptor to agonists rather than focusing on the presence of high concentrations of agonist.     

Acetylcholine release from the rabbit retina mediated by NMDA receptors.

The cholinergic amacrine cells of the rabbit retina may be labeled with 3H-choline, and the activity of the cholinergic population may be monitored by following the release of 3H-ACh. In magnesium-free medium, the glutamate analog NMDA caused massive ACh release, up to 50x the basal efflux. Magnesium blocked the NMDA-evoked release of ACh with an IC50 of 151 microM. The NMDA-evoked release of ACh was unchanged in glycine-free medium or in the presence of 500 microM glycine. However, the block of NMDA-evoked release by 7-chlorokynurenic acid (7-Cl-Kyn) was reversed by exogenous glycine. This suggests that the NMDA receptors mediating ACh release possess an allosteric glycine binding site, but under normal conditions, it is saturated by endogenous glycine. Submaximal doses of NMDA were used to determine the potency of NMDA antagonists and their specificity was established with submaximal doses of other glutamate agonists. DL-2-amino-7-phosphonoheptanoate (DL-AP-7) was a competitive NMDA antagonist, with an IC50 of 33 microM and (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) was a noncompetitive NMDA antagonist, with an IC50 of 10.6 nM. Neither antagonist blocked the light-evoked release of ACh from the retina. Furthermore, light stimulation did not activate the use-dependent block characteristic of MK-801, indicating that the endogenous transmitter did not open the NMDA channel. These results suggest that NMDA receptors do not mediate the physiological input to cholinergic amacrine cells in the rabbit retina.     

Presynaptic GABAB receptor activation attenuates synaptic transmission to rat sympathetic preganglionic neurons in vitro.

Intracellular recordings were made from sympathetic preganglionic neurons (SPNs) in transverse neonate rat spinal cord slices. Superfusion of gamma-aminobutyric acid (GABA; 25-100 microM) or (-)-baclofen (1-30 microM) consistently attenuated the excitatory postsynaptic potentials (EPSPs) evoked by stimulation of dorsal rootlets or lateral funiculus, without causing a significant change of the resting membrane potential and input resistance of the SPNs or of the depolarizations induced by pressure applications of glutamate; the IC50 for baclofen was 2.5 microM. When superfused at a higher concentration (greater than or equal to 500 microM) or ejected by pressure GABA caused a bicuculline-sensitive membrane hyperpolarization. The enantiomer (+)-baclofen (10-50 microM) and the GABAA agonist muscimol (1-10 microM) had no significant effect on the EPSPs. The GABAB receptor antagonist 2-hydroxy-saclofen caused a 10 fold rightward shift of the baclofen dose-response curve, whereas the GABAA receptor antagonist bicuculline (10-50 microM) was ineffective. Glycine had no significant effects on the EPSPs in the concentrations (10-100 microM) tested here. The results indicate that of the two putative inhibitory transmitters in the spinal cord GABA but not glycine depresses EPSPs evoked in the rat SPNs by acting on presynaptic GABAB receptors, the activation of which results in a reduction of excitatory transmitter release.     

Adenosine inhibits L-type Ca2+ current and catecholamine release in the rabbit carotid body chemoreceptor cells

In an in vitro preparation of the intact carotid body (CB) of the rabbit, adenosine (100 microM) inhibited hypoxia-induced catecholamine release by 25%. The specific A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 microM) prevented the inhibition and increased the response to hypoxia further. In isolated chemoreceptor cells from the same species, adenosine inhibited voltage-dependent Ca2+ currents by 29% at 1 microM (concentration producing half-maximal inhibition, IC50 = 50 nM). This inhibition was mimicked by R(-)N6-(2-phenylisopropyl)-adenosine and 2-chloroadenosine (1 microM), two purinergic agonists poorly active at the intracellular ('P') site, and persisted in the presence of dipyridamole (a blocker of adenosine uptake; 1 microM) and was fully inhibited by 8-phenyltheophylline (10 microM). The A1 antagonists DPCPX (10 microM) and 8-cyclopentyl-1,3-dimethylxantine (0.1 microM) inhibited the effect of adenosine by 93% (IC50 = 0.14 microM) and 59%, respectively. The inhibition of the Ca2+ current (I(Ca)) was reduced by nisoldipine (an L-type Ca2+ channel antagonist) by nearly 50%, and was unaltered by omega-conotoxin GVIA, a blocker of N-type Ca2+ channels. Adenosine did not affect the voltage-dependent Na+ current (I(Na)) or K+ current (I(K)). We conclude that adenosine A1 receptors are located in chemoreceptor cells and mediate the inhibition of L-type Ca2+ channels and thereby the release of catecholamines produced by hypoxia. The data also indicate that endogenous adenosine acts as a physiological negative modulator of the chemoreceptor cell function. The previously reported excitatory action of adenosine on the activity of the sensory nerve of the CB is discussed in terms of a balance between the inhibition mediated by A1 receptors and the excitation mediated by A2 receptors.     

Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists

The antagonist pharmacology of glutamate neurotoxicity was quantitatively examined in murine cortical cell cultures. Addition of 1-3 mM DL-2-amino-5-phosphonovalerate (APV), or its active isomer D-APV, acutely to the exposure solution selectively blocked the neuroexcitation and neuronal cell selectively blocked the neuroexcitation and neuronal cell loss produced by N-methyl-D-aspartate (NMDA), with relatively little effect on that produced by either kainate or quisqualate. As expected, this selective NMDA receptor blockade only partially reduced the neuroexcitation or acute neuronal swelling produced by the broad-spectrum agonist glutamate; surprisingly, however, this blockade was sufficient to reduce glutamate-induced neuronal cell loss markedly. Lower concentrations of APV or D-APV had much less protective effect, suggesting that the blockade of a large number of NMDA receptors was required to acutely antagonize glutamate neurotoxicity. This requirement may be caused by the amplification of small amounts of acute glutamate-induced injury by subsequent release of endogenous NMDA agonists from injured neurons, as the "late" addition of 10-1000 microM APV or D-APV (after termination of glutamate exposure) also reduced resultant neuronal damage. If APV or D-APV were present both during and after glutamate exposure, a summation dose-protection relationship was obtained, showing substantial protective efficacy at low micromolar antagonist concentrations. Screening of several other excitatory amino acid antagonists confirmed that the ability to antagonize glutamate neurotoxicity might correlate with ability to block NMDA-induced neuroexcitation: The reported NMDA antagonists ketamine and DL-2-amino-7-phosphono-heptanoate, as well as the broad-spectrum antagonist kynurenate, were all found to attenuate glutamate neurotoxicity substantially; whereas gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds reported to block predominantly quisqualate or kainate receptors, did not affect glutamate neurotoxicity. The present study suggests that glutamate neurotoxicity may be predominantly mediated by the activation of the NMDA subclass of glutamate receptors--occurring both directly, during exposure to exogenous compound, and indirectly, due to the subsequent release of endogenous NMDA agonists. Given other studies linking NMDA receptors to channels with unusually high calcium permeability, this suggestion is consistent with previous data showing that glutamate neurotoxicity depends heavily on extracellular calcium.     

Bicuculline- and Phaclofen-Resistant Hyperpolarizations Evoked by Glutamate Applications to Stratum Lacunosum-Moleculare in CA1 Pyramidal Cells of the Rat Hippocampus In Vitro

In the hippocampus, different types of interneurons may mediate distinct gamma-aminobutyric acid (GABA) responses, i.e. the early and late inhibitory postsynaptic potentials (IPSPs). To verify this hypothesis, intracellular recordings were obtained from CA1 pyramidal cells (n=63) in rat hippocampal slices. Glutamate (1 mM) was locally ejected in stratum lacunosum-moleculare to activate interneurons in this region. Glutamate-evoked hyperpolarizing responses were characterized in pyramidal cells and compared to the early IPSP and the late IPSP elicited by stratum radiatum electrical stimulation. Several characteristics were similar for the glutamate-evoked IPSPs and late IPSPs: their amplitude was small (-3.4 versus -4.9 mV, respectively), each was associated with a small conductance increase (5.0 versus 9.3 nS, respectively), their peak latency was slow (124.4 versus 129.8 ms, respectively) and in the majority of cells, each displayed little response reversal. However, the equilibrium potential of the glutamate IPSP (-76.5 mV) was similar to that of the early IPSP (-73.8 mV). Perfusion with a low Ca2+ (0.5 mM)/high Mg2+ (8 mM) medium or with tetrodotoxin (1 microM), which blocked synaptic transmission, also reduced the glutamate IPSP. Therefore the glutamate IPSP may be mediated indirectly by inhibitory interneurons. The GABAA antagonist bicuculline (10 microM), or picrotoxin (10-20 microM), blocked the early IPSP, but not the glutamate IPSP. The GABAB antagonist phaclofen (1 mM) attenuated the late IPSP, but did not affect the glutamate IPSP. The results of these experiments suggest that glutamate stimulation of interneurons in stratum lacunosum-moleculare evokes a slow IPSP different from the GABA-mediated early and late IPSPs in CA1 pyramidal cells of the hippocampus.     

Glutamate‐induced depression of EPSP–spike coupling in rat hippocampal CA1 neurons and modulation by adenosine receptors

The presence of high concentrations of glutamate in the extracellular fluid following brain trauma or ischaemia may contribute substantially to subsequent impairments of neuronal function. In this study, glutamate was applied to hippocampal slices for several minutes, producing over‐depolarization, which was reflected in an initial loss of evoked population potential size in the CA1 region. Orthodromic population spikes recovered only partially over the following 60 min, whereas antidromic spikes and excitatory postsynaptic potentials (EPSPs) showed greater recovery, implying a change in EPSP–spike coupling (E–S coupling), which was confirmed by intracellular recording from CA1 pyramidal cells. The recovery of EPSPs was enhanced further by dizocilpine, suggesting that the long‐lasting glutamate‐induced change in E–S coupling involves NMDA receptors. This was supported by experiments showing that when isolated NMDA‐receptor‐mediated EPSPs were studied in isolation, there was only partial recovery following glutamate, unlike the composite EPSPs. The recovery of orthodromic population spikes and NMDA‐receptor‐mediated EPSPs following glutamate was enhanced by the adenosine A1 receptor blocker DPCPX, the A_2A receptor antagonist SCH58261 or adenosine deaminase, associated with a loss of restoration to normal of the glutamate‐induced E–S depression. The results indicate that the long‐lasting depression of neuronal excitability following recovery from glutamate is associated with a depression of E–S coupling. This effect is partly dependent on activation of NMDA receptors, which modify adenosine release or the sensitivity of adenosine receptors. The results may have implications for the use of A₁ and A_2A receptor ligands as cognitive enhancers or neuroprotectants.     

Inhibition of synaptically evoked cortical acetylcholine release by intracortical glutamate: involvement of GABAergic neurons

Cortical acetylcholine (ACh) has been shown to regulate diverse cognitive processes and its release can be regulated by neuromodulators that act presynaptically at cholinergic terminals. The neocortex receives dense glutamatergic input from thalamocortical and other fibres. The present study used in vivo microdialysis to examine, and pharmacologically characterize, the effect of glutamate on cortical ACh release evoked by electrical stimulation of the pedunculopontine tegmental nucleus in urethane-anaesthetized rats. All drugs were administered locally within the cortex by reverse dialysis. Application of glutamate had no detectable effect on spontaneous ACh release but reduced evoked cortical ACh efflux in a concentration-dependent manner. This effect was mimicked by the glutamate transporter blocker L-trans-pyrrolidine-2,4-dicarboxylic acid, as well as by the ionotropic glutamate receptor agonists N-methyl-D-aspartic acid and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and was blocked by the ionotropic glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and (+/-)-3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid. Glutamate application also increased extracellular adenosine levels but the simultaneous delivery of the broad-spectrum adenosine receptor antagonist caffeine failed to affect the inhibitory action of glutamate on evoked ACh release. However, the effect of glutamate was fully blocked by simultaneous delivery of the GABAA receptor antagonist bicuculline and partially blocked by the GABAB receptor antagonist phaclofen. These results suggest that ionotropic glutamate receptor activation by glutamate inhibits evoked cortical ACh release via an indirect pathway involving GABAergic neurons in the cortex.     

Presynaptic inhibition of excitatory synaptic transmission by adenosine in rat hippocampus: analysis of unitary EPSP variance measured by whole-cell recording.

We have utilized the favorable signal-to-noise ratios provided by whole-cell recording, combined with variance analysis, to determine the pre- or postsynaptic actions of a variety of manipulations on unitary EPSPs evoked by low-intensity stimulation of afferents to CA1 pyramidal neurons in slices of hippocampus. Estimates of quantal content (mcv) were determined by calculating the ratio of the squared average unitary EPSP amplitude (determined from 150-275 responses) to the variance of these responses (M2/sigma 2), while quantal amplitudes (qcv) were estimated by calculating the ratio of the response variance to average EPSP size (sigma 2/M). Estimates of mcv were highly correlated with those determined using the method of failures (mf). With paired stimulation (50 msec interpulse interval) there was a significant facilitation of the second unitary EPSP, accompanied by an increase in mcv, but not qcv, suggesting that this facilitation was of presynaptic origin. Superfusion of hippocampal slices with various concentrations of adenosine, the A1-selective adenosine receptor agonist cyclohexyladenosine, or the Ca2+ channel blocker cadmium significantly reduced average unitary EPSP amplitudes and mcv, without significantly altering qcv, suggesting a presynaptic locus for this inhibition. The 50% effective concentration for the apparent presynaptic action of adenosine on mcv in the present study (5.7 microM; 95% confidence limits = 4.2-7.7 microM) was significantly lower than its EC50 for reducing conventional, large EPSPs (33 microM; recorded with high-resistance microelectrodes), or extracellular field EPSPs (29 microM), as previously reported by this laboratory. The glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) reduced average unitary EPSP amplitudes; in contrast to the above manipulations, it had no effect on mcv, but significantly altered qcv, which is consistent with its presumed postsynaptic mechanism of action. We conclude from these data that adenosine presynaptically reduces synaptic strength at Schaffer collateral-commissural synapses in the hippocampus by diminishing the number of quanta released, not by reducing the size of these individual quanta or postsynaptic sensitivity to excitatory neurotransmitter. These results suggest that the mechanism by which adenosine inhibits synaptic transmission in the hippocampus is similar, if not identical, to the mechanism by which it inhibits synaptic transmission at the neuromuscular junction.     

The A3 adenosine receptor agonist 2-Cl-IB-MECA facilitates epileptiform discharges in the CA3 area of immature rat hippocampal slices

The effects of the A(3) adenosine receptor agonist 2-Cl-IB-MECA were tested on epileptiform field potentials recorded in the CA3 area of postnatal days 10-20 immature hippocampal slices, during perfusion with the GABA(A) receptor antagonist bicuculline (10 microM). Evoked potentials: 2-Cl-IB-MECA (1-50 microM, n = 17) had consistently excitatory effects, blocked by the A(3) receptor antagonist MRS 1220 (1 microM, n = 7), but not occluded in the presence of the A(1) antagonist DPCPX (1 microM, n = 12) or the A(2A) antagonist ZM-241385 (0.1 microM, n = 12). 2-Cl-IB-MECA reversed the inhibitory effects (n = 5) of the adenosine uptake blocker nitrobenzylthioinosine (NBTI, 50 microM), but did not increase its excitatory effects (n = 19). Spontaneous discharges: 2-Cl-IB-MECA (1 microM) induced them or increased their frequency in 14/30 slices, an effect reversed by MRS 1220 (n = 3), and observed also following pre-perfusion with DPCPX (n = 11), ZM-241385 (n = 11) or both (n = 10). In the presence of the A(1) antagonist DPCPX, NBTI increased the frequency of spontaneous discharges, an effect partially reversed by MRS 1220 (n = 8), thus suggesting that a rise in endogenous adenosine during disinhibition may activate A(3) receptors. In conclusion, these findings suggest strongly that activation of A(3) receptors, following a rise in endogenous adenosine (i.e. during seizures, hypoxia), facilitates excitation, thus limiting the known inhibitory and/or neuroprotective effects of adenosine in immature brain.     

Adenosine A1 receptor-mediated depression of corticostriatal and thalamostriatal glutamatergic synaptic potentials in vitro

Electrophysiological recordings in rat brain slices have been used to study the actions of adenosine on striatal neurons and striatal excitatory amino acid neurotransmission originating in the cortex or the thalamus. Adenosine had no effects on membrane properties of striatal neurons. Adenosine and the A₁ agonist N⁶-Cyclopentyl adenosine reduced EPSPs of both cortical and thalamic origin by more than 50%. Depression of EPSPs was associated with an increase in paired-pulse facilitation, suggesting a presynaptic locus of action. EPSP depression was blocked by the A₁ antagonist, 8-Cyclopentyl-1,3-dipropyl xanthine. The A₂ agonist 5′-(N-cyclopropyl)-carboxamidoadenosine had no effect on excitatory amino acid neurotransmission. The A₁ antagonist alone enhanced the synaptic component of the evoked field potential (23±12%). These results indicate that endogenous adenosine, acting via A₁ receptors, limits striatal glutamatergic neurotransmission, serving a modulatory and neuroprotective role.     

Electrophysiological actions of acetate, a metabolite of ethanol, on hippocampal dentate granule neurons: interactions with adenosine.

Acetate is the primary breakdown product of ethanol metabolism in the liver and has been found in the brain following ethanol ingestion in rats. Systemically administered acetate has been shown to cause motor impairment, an effect which is blocked by the adenosine receptor blocker, 8-phenyltheophylline (8-PT). The effects of sodium acetate were investigated in this study using intracellular recording techniques in rat hippocampal dentate granule cells, and were compared to the actions of ethanol and adenosine individually and in conjunction with 8-PT. Acetate hyperpolarized the membrane at 0.4-0.8 mM. The amplitude and duration of the postspike train afterhyperpolarization (AHP) were increased by acetate when the cell was repolarized to the control resting membrane potential. Comparable results were seen in voltage clamp. Acetate also decreased spike frequency adaptation. The effects of acetate were mimicked by adenosine (50 microM) and ethanol (20 mM). The ethanol effects occluded those produced by acetate. All of the effects of acetate, adenosine and ethanol could be inhibited with prior perfusion of 8-PT (1-10 microM). These data suggest that the actions of the major metabolite of ethanol, acetate, may be mediated by adenosine receptor activation.