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The second coding exon of the adenosine A, receptor gene was eliminated by homologous recombination. The phenotype of mice (mixed C57B6/129OlaHsd background) was studied, using siblings from matings of heterozygous mice. Among the offspring the ratio between+/+, +/-and -/-animals was 1:2:1. Over the first half-year-at least-growth and viability were the same in all genotypes. Binding of A(1) ligands was eliminated in-/-mice and halved in+/-mice. Blood pressure was increased in-/-mice and this was paralleled by an increase in plasma renin. Heart rate was unaffected, as was contractility. Furthermore, the response of the perfused heart to ischemia was similar in+/+and -/-hearts. However, remote preconditioning was eliminated in-/-mouse hearts. Tubuloglomerular feedback in the kidney was also lost in-/-mice. The analgesic response to a non-selective adenosing receptor agonist was lost in-/-mice, which also showed hyperalgesia in the tail-flick test. There was a slight hypoactivity in-/-mice, but responses to caffeine were essentially normal. The inhibition of excitatory neurotransmission in hippocampus by adenosine was lost in-/-mice and reduced in+/-mice. Responses to ATP were affected similarly. Hypoxic depression of synaptic transmission was essentially eliminated in hippocampus and hypoxic decrease in spinal respiratory neuron firing was markedly reduced. These results show that adenosine A, receptors play a physiologically important role in the kidney, spinal cord, and hippocampus and that they are critically important in the adaptive responses to hypoxia. (C) 2003 Wiley-Liss, Inc. (Less)

Ethanol intoxication results partly from actions of ethanol at specific ligand-gated ion channels. One such channel is the GABA A receptor complex, although ethanol9s effects on GABA A receptors are variable. For example, we found that hippocampal neurons from selectively bred mice and rats with high hypnotic sensitivity to ethanol have increased GABA A receptor-mediated synaptic responses during acute ethanol treatment compared with mice and rats that display low behavioral sensitivity to ethanol. Here we investigate whether specific protein kinase C (PKC) isozymes modulate hypnotic and GABA A receptor sensitivity to ethanol. We examined acute effects of ethanol on GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs) in mice lacking either PKCγ (PKCγ −/− ) or PKCe (PKCe −/− ) isozymes and compared the results to those from corresponding wild-type littermates (PKCγ +/+ and PKCe +/+ ). GABA A receptor-mediated IPSCs were evoked in CA1 pyramidal neurons by electrical stimulation in stratum pyramidale, and the responses were recorded in voltage-clamp mode using whole-cell patch recording techniques. Ethanol (80 mM) enhanced the IPSC response amplitude and area in PKCγ +/+ mice, but not in the PKCγ −/− mice. In contrast, ethanol markedly potentiated IPSCs in the PKCe −/− mice, but not in PKCe +/+ littermates. There was a positive correlation between ethanol potentiation of IPSCs and the ethanol-induced loss of righting reflex such that mice with larger ethanol-induced increases in GABA A receptor-mediated IPSCs also had higher hypnotic sensitivity to ethanol. These results suggest that PKCγ and PKCe signaling pathways reciprocally modulate both ethanol enhancement of GABA A receptor function and hypnotic sensitivity to ethanol.

The ability of ethanol to enhance GABA(A) receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABA(A) inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11-15 degrees C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABA(A) IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31-33 degrees C. In addition, the increase in ethanol sensitivity of GABA(A) IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABA(A) receptor-mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABA(A) receptors in the mammalian brain.

Background Nicotine and alcohol are both drugs of abuse that are frequently used concurrently, but little is known about the interactions between these agents at the molecular level. The neuronal subgroup of nicotinic acetylcholine receptors (nAChRs) are members of a neurotransmitter-gated ion channel superfamily and have been used extensively to characterize the interactions of ethanol with these channels. Previous work has shown that both the direction and the magnitude of the modulatory effects of n-alcohols and several fluorinated analogs on nAChRs can be predicted based upon the molecular volume of the alkanol. However, size (molecular volume) is only one of several determinants of binding-site interactions; shape and flexibility of the interacting ligand and/or the site itself (i.e., structural constraints) and alkanol hydrophobicity are also likely to be involved. Methods Two-electrode voltage-clamp electrophysiology was used to investigate the effects of alkanols on the alpha2beta4 human neuronal nicotinic acetylcholine receptor ectopically expressed in Xenopus oocytes. Results ACh-induced currents mediated by alpha2beta4 nAChRs were potentiated by 1- and 2-propanol, whereas 1-pentanol and several branched-chain pentanol isomers only inhibited channel activity. 1-Butanol had a biphasic concentration-response curve, producing a nearly 2-fold increase in current at lower concentrations and >50% inhibition at higher concentrations. Other four-carbon alcohols such as 2-butanol and 2-methyl-1-propanol also facilitated responses at lower concentrations and inhibited at higher concentrations, whereas 2-butene-1-ol and 2-methyl-2-propanol only inhibited responses. The effects of these alkanols on ACh responses were the result of changes in both the potency as well as the efficacy of ACh at these receptors. Conclusions Although molecular volume and hydrophobicity both correlate well with the potencies of linear alkanols in modulating the activity of alpha2beta4 nAChRs, these relationships break down almost completely when comparing the structural isomers of these agents. The structure of the interacting alkanol significantly affects the potency and efficacy of alkanols at the nAChR, and this occurs independently of differences in molecular volume and hydrophobicity.

Caffeine is believed to act by blocking adenosine A1and A2Areceptors (A1R, A2AR), indicating that some A1receptors are tonically activated. We generated mice with a targeted disruption of the second coding exon of the A1R (A1R−/−). These animals bred and gained weight normally and had a normal heart rate, blood pressure, and body temperature. In most behavioral tests they were similar to A1R+/+mice, but A1R−/−mice showed signs of increased anxiety. Electrophysiological recordings from hippocampal slices revealed that both adenosine-mediated inhibition and theophylline-mediated augmentation of excitatory glutamatergic neurotransmission were abolished in A1R−/−mice. In A1R+/−mice the potency of adenosine was halved, as was the number of A1R. In A1R−/−mice, the analgesic effect of intrathecal adenosine was lost, and thermal hyperalgesia was observed, but the analgesic effect of morphine was intact. The decrease in neuronal activity upon hypoxia was reduced both in hippocampal slices and in brainstem, and functional recovery after hypoxia was attenuated. Thus A1Rs do not play an essential role during development, and although they significantly influence synaptic activity, they play a nonessential role in normal physiology. However, under pathophysiological conditions, including noxious stimulation and oxygen deficiency, they are important.

▪ Abstract Adenosine is a modulator that has a pervasive and generally inhibitory effect on neuronal activity. Tonic activation of adenosine receptors by adenosine that is normally present in the extracellular space in brain tissue leads to inhibitory effects that appear to be mediated by both adenosine A1and A2Areceptors. Relief from this tonic inhibition by receptor antagonists such as caffeine accounts for the excitatory actions of these agents. Characterization of the effects of adenosine receptor agonists and antagonists has led to numerous hypotheses concerning the role of this nucleoside. Previous work has established a role for adenosine in a diverse array of neural phenomena, which include regulation of sleep and the level of arousal, neuroprotection, regulation of seizure susceptibility, locomotor effects, analgesia, mediation of the effects of ethanol, and chronic drug use.

Although it has been demonstrated repeatedly that ATP is a fast excitatory transmitter in the central nervous system (CNS), there are only limited data to indicate a corelease of ATP with other transmitters such as γ-aminobutyric acid or noradrenaline. Somewhat surprisingly, there is no evidence hitherto for glutamate-ATP cotransmission in spite of the widespread distribution of glutamatergic neurons in the brain and spinal cord. However, ATPergic and glutamatergic neurons may interact both at the pre- and postsynaptic level. Presynaptic P2X receptors facilitate glutamate release both from the terminals of mesencephalic proprioceptive trigeminal neurons projecting to the motor trigeminal nucleus in the brainstem and from primary afferent fibers onto dorsal horn neurons of the spinal cord. The inhibitory effect of ATP via presynaptic P2Y receptors has been demonstrated convincingly at noradrenergic but not glutamatergic nerve terminals. However, in addition to the direct effects of ATP, adenosine formed by the enzymatic degradation of ATP can inhibit the release of glutamate from the Schaffer collateral-commissural patway onto CA1 pyramidal cells of the hippocampus. Similarly, ATP degraded to adenosine may inhibit the N-methyl-D-aspartate (NMDA)-induced current in the striatopallidal subpopulation of medium spiny neurons via A 2A receptor activation. Finally, ATP may potentiate the NMDA receptor-mediated depolarization or the underlying inward current at layer V pyramidal neurons of the prefrontal cortex, and P2Y receptors have been suggested to mediate this interaction. Thus, although as a transmitter ATP alters the membrane potential of a limited subset of CNS neurons, it may have widespread and pronounced effects by modulating glutamatergic mechanisms.

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.

Prolonged incubation of hippocampal brain slices at reduced temperature immediately after preparation (cold shock treatment) has been shown to lead to an extremely persistent activation of protein kinase C (PKC). Because of the numerous suggestions that adenosine receptor sensitivity can be modulated by PKC, we have characterized the changes in both pre- and postsynaptic adenosine A 1 receptor sensitivity following such treatment. The maximal effect of activation of postsynaptic A 1 receptors was significantly reduced, although there was no change in the EC 50 value. On the other hand, the sensitivity of presynaptic adenosine receptors was slightly although significantly increased (the EC 50 value was reduced), with no apparent change in the maximal effect. Finally, the effect of dipyridamole was reduced in these slices, suggesting that its ability to inhibit adenosine transport was reduced. These results suggest that the cold shock treatment does not affect PKC activity in all cellular compartments and that, in particular, PKC-dependent changes in nerve terminal and somatic A 1 receptors are markedly different.

The vagal lobe in goldfish is a laminated structure in the midmedulla responsible for processing vagal gustatory input from the oropharynx. The anatomical arrangement of the vagal lobe is conducive to an in vitro slice preparation for investigating the physiology and pharmacology of primary gustatory fibers. Postsynaptic population responses (N2 and N3) were evoked from sensory layers of the vagal lobe following stimulation of the incoming vagal fibers. Application of 100 microM kynurenic acid, a broad spectrum glutamate receptor antagonist, abolished or significantly decreased the evoked responses. These results indicate that excitatory amino acids are the neurotransmitter at the first relay in the taste pathway in the central nervous system.

Exogenous application of acetylcholine elicits inward currents in hippocampal interneurons that are mediated via α-bungarotoxin-sensitive nicotinic acetylcholine receptors, but synaptic responses mediated via such receptors have never been reported in mammalian brain. In the present study, EPSCs were evoked in hippocampal interneurons in rat brain slices by electrical stimulation and were recorded by using whole-cell voltage-clamp techniques. Nicotinic EPSCs were isolated pharmacologically, using antagonists to block other known types of ligand-gated ion channels, and then were tested with either α-bungarotoxin or methyllycaconitine, which are selective antagonists for nicotinic acetylcholine receptors that contain the α7 receptor subunit. Each antagonist proved highly effective at reducing the remaining synaptic current. Evoked α7-mediated nicotinic EPSCs also were desensitized by superfusion with 1 μmnicotine, had extrapolated reversal potentials near 0 mV, and showed strong inward rectification at positive potentials. In several interneurons, methyllycaconitine-sensitive spontaneous EPSCs also were observed that exhibited a biphasic decay rate very similar to that of the α7-mediated evoked response. These studies provide the first demonstration of a functional cholinergic synapse in the mammalian brain, in which the primary postsynaptic receptors are α-bungarotoxin-sensitive nicotinic acetylcholine receptors.

We examined the effects of acute ethanol exposure on recombinant human glycine receptors transiently transfected into HEK 293 cells and stably transfected into Ltk- fibroblast-like cells. In our study of the effects of ethanol, we used the whole-cell patch-clamp configuration. Relatively low concentrations of ethanol (25 mM and 50 mM) did not affect glycine-gated currents in any of the cell lines studied. Higher concentrations of ethanol (100 mM and 200 mM) significantly potentiated glycine responses only in stably transfected Ltk- cells expressing alpha1 and alpha2 subunits and in HEK 293 cells transiently expressing alpha2 subunits. Cells stably expressing alpha1 versus alpha2 glycine receptors were modulated equally by ethanol. Both glycine alpha1 and glycine alpha1beta receptors transiently expressed in HEK 293 cells were insensitive to all concentrations of ethanol tested; however, there was a trend toward potentiation at 100 and 200 mM ethanol concentrations. A population of cells (41-87%) that was sensitive to the potentiating effects of 100 and 200 mM ethanol (defined as more than 10% potentiation) was identified in both cell lines tested. In these sensitive cells, ethanol (100 and 200 mM) produced significant potentiation, independent of the cell line and the glycine receptor subunit tested. Together with published results from studies with Xenopus oocytes, these data indicate that the sensitivity of recombinant glycine receptors to ethanol depends upon the expression system.

Extracellular adenosine 3':5'-cyclic monophosphate (cAMP) is a potential source of the inhibitory neuromodulator adenosine in the brain. Previous work has demonstrated that cAMP, which is formed intracellularly, can be transported into the extracellular space and subsequently catabolized to adenosine. However, the physiological conditions under which cAMP release might lead to adenosine formation and activation of adenosine receptors are not well understood. In this study we demonstrate that superfusion of hippocampal slices with cAMP or forskolin led to the formation of extracellular adenosine which activated adenosine receptors in a manner comparable to that seen with adenosine superfusion. In contrast, application of brief pulses of cAMP onto the cell bodies of CA1 pyramidal neurons failed to produce an adenosine receptor-mediated response, while application of brief pulses of adenosine or AMP elicited significant responses. These data suggest that large, prolonged increases in extracellular cAMP levels can result in the formation of extracellular adenosine and the activation of adenosine receptors, but brief increases in cAMP levels in the vicinity of individual neurons cannot. These findings imply that increases in cAMP levels may lead to relatively slow increases in extracellular adenosine, as opposed to the fast, spatially restricted increases that would occur following the release of other adenine nucleotides.

Weiner, J. L., C. Gu, and T. V. Dunwiddie. Differential ethanol sensitivity of subpopulations of GABAA synapses onto rat hippocampal CA1 pyramidal neurons. J. Neurophysiol. 77: 1306–1312, 1997. The actions of ethanol on γ-aminobutyric acid-A (GABAA) receptor-mediated synaptic transmission in rat hippocampal CA1 neurons remain controversial. Recent studies have reported that intoxicating concentrations of ethanol (10–100 mM) can potentiate, inhibit, or have no effect on GABAA receptor-mediated synaptic responses in this brain region. The essential determinants of ethanol sensitivity have not been defined; however, GABAA receptor subunit composition, as well as posttranslational modifications of these receptors, have been suggested as important factors in conferring ethanol sensitivity to the GABAA receptor complex. Multiple types of GABAA receptor-mediated synaptic responses have been described within individual hippocampal CA1 neurons. These responses have been shown to differ in some of their physiological and pharmacological properties. In the present study we tested the hypothesis that some of the disparate findings concerning the effects of ethanol may have resulted from differences in the ethanol sensitivity of GABAA receptor-mediated synapses on single CA1 pyramidal cells. Electrical stimulation adjacent to the stratum pyramidale (proximal) and within the stratum lacunosum-moleculare (distal) activated nonoverlapping populations of GABAA receptors on rat hippocampal CA1 neurons. Proximal inhibitory postsynaptic currents (IPSCs) decayed with a single time constant and were significantly potentiated by ethanol at all concentrations tested (40, 80, and 160 mM). Distal IPSCs had slower decay rates that were often described better by the sum of two exponentials and were significantly less sensitive to ethanol at all concentrations tested. Three other allosteric modulators of GABAA receptor function with well-defined GABAA receptor subunit requirements, pentobarbital, flunitrazepam, and zolpidem, potentiated proximal and distal GABAA IPSCs to the same extent. These results demonstrate that the ethanol sensitivity of GABAA receptors can differ, not only between brain regions but within single neurons. These findings offer a possible explanation for the conflicting results of previous studies on ethanol modulation of GABAA receptor-mediated synaptic transmission in rat hippocampal CA1 neurons.

The adenosine A3receptor is expressed in brain, but the consequences of activation of this receptor on electrophysiological activity are unknown. We have characterized the actions of a selective adenosine A3receptor agonist, 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA), and a selective A3receptor antagonist, 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191), in brain slices from rat hippocampus. In the CA1 region, activation of A3receptors had no direct effects on synaptically evoked excitatory responses, long-term potentiation, or synaptic facilitation. However, activation of A3receptors with Cl-IB-MECA antagonized the adenosine A1receptor-mediated inhibition of excitatory neurotransmission. The effects of Cl-IB-MECA were blocked by pretreatment with MRS 1191, which by itself had no effect on A1receptor-mediated responses. The presynaptic inhibitory effects of baclofen and carbachol, mediated via GABABand muscarinic receptors, respectively, were unaffected by Cl-IB-MECA. The maximal response to adenosine was unchanged, suggesting that the primary effect of Cl-IB-MECA was to reduce the affinity of adenosine for the receptor rather than to uncouple it. Similar effects could be demonstrated after brief superfusion with high concentrations of adenosine itself. Under normal conditions, endogenous adenosine in brain is unlikely to affect the sensitivity of A1receptors via this mechanism. However, when brain concentrations of adenosine are elevated (e.g., during hypoxia, ischemia, or seizures), activation of A3receptors and subsequent heterologous desensitization of A1receptors could occur, which might limit the cerebroprotective effects of adenosine under these conditions.

New exciting developments on the occurrence and functional role of purinoceptors in mammalian brain were presented at the session Purinoceptors in the central nervous system chaired by Flaminio Cattabeni and Tom Dunwiddie at the Purines '96 international conference. The focus of the session were topics of recent interest, including the sources and mechanisms involved in ATP and adenosine release during physiological neurotransmission in hippocampus, the brain expression of the recently cloned P2 receptors, and the role of the various adenosine receptor subtypes in brain protection from neurodegeneration associated with trauma-, ischemia- and excessive excitatory amino acid neurotransmission. New important insights into the mechanisms responsible for the formation and release of adenosine into the extracellular space were provided by data obtained by Dunwiddie and coworkers in hippocampal pyramidal neurons. These data may have functional implications for the role of purines in modulation of synaptic plasticity and long-term potentiation in this brain area, and hence in cognitive functions. Buell provided an updated overview on the cloning, molecular characteristics and brain expression of various ligand-gated P2X purinoceptors; although the functional role of these receptors in mammalian brain still awaits elucidation, their widespread distribution in the nervous system strongly suggests that ATP-mediated events are more prevalent and important in brain than expected. Pedata presented data on the functional interrelationships between adenosine and glutamate in the brain, and also provided evidence for alterations of the reciprocal regulation between these two systems in aged brain, which may have important implications for both ischemia- and trauma-associated neurodegenerative events and senescence-associated cognitive impairment. Finally, von Lubitz provided novel data on the molecular mechanisms likely to be at the basis of the brain protective effects associated with the chronic stimulation of the adenosine A 3 receptor, further confirming that this receptor represents a crucial target for the development of new antiischemic and antineurodegenerative therapeutic agents.

Adenosine is a potent neuromodulator in the CNS, but the mechanisms that regulate adenosine concentrations in the extracellular space remain unclear. The present study demonstrates that increasing the intracellular concentration of adenosine in a single hippocampal CA1 pyramidal neuron selectively inhibits the excitatory postsynaptic potentials in that cell. Loading neurons with high concentrations of adenosine via the whole-cell patch-clamp technique did not affect the GABAA-mediated inhibitory postsynaptic potentials, the membrane resistance, or the holding current, whereas it significantly increased the adenosine receptor-mediated depression of excitatory postsynaptic currents. The effects of adenosine could not be mimicked by an agonist at the intracellular adenosine P-site, but the effects could be antagonized by a charged adenosine receptor antagonist and by adenosine deaminase, demonstrating that the effect was mediated via adenosine acting at extracellular adenosine receptors. The effect of adenosine loading was not blocked by BaCl2and therefore was not caused by an adenosine-activated postsynaptic potassium conductance. Adenosine loading increased the paired-pulse facilitation ratio, demonstrating that the effect was mediated by presynaptic adenosine receptors. Finally, simultaneous extracellular field recordings demonstrated that the increase in extracellular adenosine was confined to excitatory synaptic inputs to the loaded cell. These data demonstrate that elevating the intracellular concentration of adenosine in a single CA1 pyramidal neuron induces the release of adenosine into the extracellular space in such a way that it selectively inhibits the excitatory inputs to that cell, and the data support the general conclusion that adenosine is a retrograde messenger used by pyramidal neurons to regulate their excitatory input.

Platelet-derived growth factor (PDGF) is a multifunctional protein that plays important roles in many tissues, including the mammalian central nervous system. PDGF and PDGF receptors (PDGFRs) are expressed in virtually every region of the central nervous system where they are involved in the development, survival, growth, and differentiation of both neuronal and glial cells. We now report that a brief activation of PDGFRs produced a long-lasting inhibition of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents in CA1 pyramidal neurons in rat hippocampal slices. PDGF also inhibited NMDA receptors (NMDA-Rs) in cultured hippocampal neurons by a mechanism that involves a decrease in single channel open probability. Non-NMDA receptor function was not affected by PDGF in hippocampal neurons. Experiments with mutant PDGFRs and chelation of intracellular Ca2+ in Xenopus oocytes indicate that this inhibition depends on a phospholipase C-γ-induced elevation of intracellular Ca2+ levels. The PDGF-induced inhibition of NMDA-Rs is produced by a mechanism different than the well characterized phenomenon of Ca2+-dependent NMDA-R run down because the effect of PDGF was blocked by the phosphatase inhibitor, calyculin A, and was not affected by the microtubule polymerizing agent, phalloidin. Because elevations of PDGF levels are associated with neurological trauma or disease, we propose that PDGF can exert neuroprotective effects by inhibiting NMDA-R-dependent excitotoxicity.

Acetate is the primary product of ethanol catabolism and can accumulate in the blood at concentration of up to 2 mM following ethanol consumption. It has been suggested that some of the pharmacological actions of ethanol are mediated via acetate, which can lead indirectly to the release of endogenous adenosine. In the present experiments this hypothesis was tested by examining the effects of exogenous sodium acetate on the physiology of hippocampal slices from rat brain. Acetate had no significant effect on intracellular responses recorded from CA1 pyramidal neurons or on extracellular field potentials evoked from the either the CA1 region or the dentate gyrus. There was also no significant differences in responses to the adenosine receptor antagonist theophylline in CA1 pyramidal neurons recorded using intracellular filling solutions containing potassium acetate, KC1, or potassium methylsulfate. These results suggest that the presence of acetate, either in the extracellular medium or within an intracellular electrode, does not induce a significant increase in adenosine receptor activation in the hippocampus.

Adenosine was found to decrease the amplitude of presynaptic fiber spikes recorded in stratum radiatum of the CA1 region of the hippocampus following stimulation of the Schaffer collateral and commissural afferents. The presynaptic fiber spike reflects currents activated in the presynaptic fibers and nerve terminals of this projection. However, the effect of adenosine was largely the result of: (a) temporal overlap between the presynaptic fiber spike and the field EPSP response and (b) an indirect effect most likely related to the hyperpolarization of CA3 neurons giving rise to the Schaffer collateral fibers. When the CA3 region was separated by a knife cut from the CA1 region and the field EPSP blocked with 6,7-dinitroquinoxaline-2,3-dione, the effect of adenosine was markedly reduced, but was still significant (4.4 ± 1.4% reduction in the amplitude of the presynaptic fiber spike). However, this action was not mimicked by the Ca2+ channel antagonist cadmium, suggesting that it did not reflect an inhibition of Ca2+ currents by adenosine. This effect was not observed in the presence of the K+ channel antagonists Ba2+ or diaminopyridine. These results suggest that adenosine may have a small hyperpolarizing effect on the presynaptic nerve terminals, but this is unlikely to be the mechanism by which adenosine modulates transmission in this brain region.

The results of several previous studies have suggested that pretreatment with adenosine can block the induction of long-term potentiation (LTP), although other studies have found no effect of adenosine on the induction of LTP. The interaction of adenosine with the induction of LTP in the rat hippocampal slice was investigated. Inhibition of synaptic responses by adenosine either prior to or immediately after high-frequency or theta-burst stimulation did not affect LTP measured after washout of the adenosine. The only conditions under which adenosine blocked the development of LTP was when it was given 3-5 minutes prior to the stimulation train. To understand how it was possible to induce LTP, during the period 1-3 minutes following adenosine when synaptic responses were virtually eliminated, evoked responses during the 100 Hz stimulation train were recorded. Although synaptic responses to low-frequency stimulation were virtually eliminated by adenosine, they reappeared during high-frequency stimulation. These results suggest that although adenosine can depress synaptic responses, an increase in neurotransmission during a high-frequency train can partially overcome this effect of adenosine, and the hypothesis that adenosine can selectively block LTP is not supported.

Previous intracellular electrophysiological studies on rat hippocampal brain slices have shown very little effect of acute ethanol application on synaptically evoked GABA A receptor-mediated responses recorded in CA1 pyramidal neurons. The present study was designed to compare the effects of ethanol on pyramidal neurons in the hippocampus and cerebral cortex. Using conventional intracellular microelectrodes (60–80 MΩ) to impale cortical neurons in brain slices, 80 mM ethanol application did not affect the membrane input impedance nor evoked EPSPs, but significantly affected the resting membrane potential (usually a 2–5 mV hyperpolarization). When stimulus-evoked GABA A -mediated IPSCs were studied using whole-cell recordings from cortical neurons voltage-clamped at depolarizing potentials, monophasic IPSCs were evoked that were blocked by bicuculline, increased by pentobarbital, and enhanced by ethanol superfusion in a dose dependent manner over the range of 20–160 mM. Hippocampal IPSCs recorded under identical conditions were not enhanced by ethanol. Parallel studies of GABA-stimulated 36 Cl − flux measurements in microsacs prepared from hippocampal, cerebral cortical and cerebellar tissue demonstrated that ethanol significantly enhanced (30–50%) 36 Cl − flux in microsacs derived from the cerebral cortex and cerebellum, but not in microsacs prepared from the hippocampus. These results demonstrate that there are clear brain region-dependent differences in the way that GABA A receptor function is altered by acute ethanol, and that these differences are apparent not only as an enhancement of responses to exogenous GABA, but also as a facilitation of the responses to endogenous GABA released from inhibitory nerve terminals during synaptic activation.

Modulation of gamma-aminobutyric acid (GABA)-mediated inhibition, and glutamate-mediated excitation by highly selective mu and delta opioid agonists was studied using intracellular recordings of CA1 pyramidal neuron synaptic responses in superfused hippocampal slices. Equimolar concentrations of the selective mu agonist, [Tyr-(D-Ala)-Gly-(N-Me-Phe)-Gly-ol]-enkephalin (DAGO), or the delta selective agonist, [D-Pen2,D-Pen5]-enkephalin (DPDPE), reversibly increased the amplitudes of excitatory post-synaptic potentials (EPSPs), evoked by Schaffer collateral/commissural stimulation, without altering the input resistance or resting membrane potential of these CA1 pyramidal neurons. The increased EPSP amplitudes resulting from superfusion with the enkephalin analogs were qualitatively similar to those caused by the GABAA receptor antagonist, bicuculline methiodide (BMI). Specific stimulation/recording protocols and micro-lesions of the slices were used to evoke relatively pure forms of recurrent and feed-forward GABA-mediated inhibitory post-synaptic potentials (IPSPs). The mu opioid agonist DAGO reduced both recurrent and feed-forward IPSPs, while the delta agonist DPDPE had no effect upon these responses. To test the hypothesis that the enhancement of pyramidal neuron EPSPs by delta (and mu) opioids was due to the reduction of an inhibitory potential that was coincident with the EPSP, DPDPE or the mu agonist, DAGO, were applied while recording monosynaptic IPSPs following the elimination of EPSPs by the glutamate receptor antagonists, D,L-2-amino-5-phosphonovalerate (APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). The mu agonist, DAGO, reversibly reduced these pharmacologically isolated IPSPs, while the delta agonist, DPDPE, had no effect upon these responses. Despite the fact that the delta agonist, DPDPE, had no effect on recurrent, feed-forward or monosynaptic evoked IPSPs, this enkephalin did reversibly reduce the frequency of spontaneously occurring IPSPs, measured using whole-cell recordings with pipettes containing 65 mM KCl. The mu agonist, DAGO, and the GABAA antagonist, BMI, similarly reduced spontaneous IPSP rates. We conclude from these data that mu and delta opioid receptor activation increases EPSPs via the reduction of a form of GABAergic inhibition that is difficult to characterize, and which may be distinct from conventional feed-forward and recurrent inhibition. Furthermore, delta opioids seem to reduce this form of GABAergic inhibition selectively, while mu opioids reduced this inhibition, and conventional feed-forward and recurrent IPSPs as well.

The vagal lobe of goldfish (Carassius auratus) is a laminated structure in which primary gustatory afferents terminate in a stereotypical pattern. Because the afferent fibers enter and distribute within the lobe in the transverse plane, the structure appeared suitable for in vitro slice electrophysiology. Slices were cut on a vibratome at 400-800 microns thickness and placed in a fresh water teleost Ringer's solution. Following a recovery period, clear population responses were recorded following electrical stimulation of the incoming fiber bundle. The later two components of this evoked waveform were eliminated by removal of the calcium from the bathing solution indicating the synaptic origin of these potentials. Further, the waveform was highly dependent on the position of the recording electrode, both in terms of laminar and tangential position. Evoked response maxima corresponded to the layers in which the primary afferent fibers terminate. In addition, the maximal evoked response was limited to a tangential distance of approximately 100 microns. The spatial restriction of the evoked waveform therefore corresponds well with the known anatomical organization of the primary gustatory afferent fibers. The evoked waveforms are sensitive to stimulus repetition rate, being facilitated by stimulus trains less than 50 ms in duration and fatigued by stimulus repetitions as slow as 1 Hz. This in vitro preparation should provide a means for investigating the physiological and pharmacological properties of primary gustatory fibers and nuclei.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Behavioral studies have implicated central dopaminergic systems, especially the ventral tegmental area of Tsai (VTA), in the mediation of the reinforcing effects of drugs of abuse such as cocaine. A brain slice preparation of the VTA was used to assess the direct effects of cocaine on the spontaneous activity of dopamine-type neurons. When superfused with 1-10 microM cocaine the firing rate of spontaneously active VTA neurons was decreased, with no corresponding change in spike height. While there was a considerable variability in the response to a given concentration of cocaine among the individual units, every cell inhibited by dopamine was also inhibited by cocaine. The local anesthetic lidocaine had variable effects on firing rate, but never potentiated the inhibitory effects of dopamine. Inhibitory responses to either dopamine or cocaine were blocked by the specific D2 dopamine receptor antagonist sulpiride. Small concentrations of cocaine (0.1-0.5 microM), which by themselves had little or no effect on spontaneous activity, potentiated the inhibitory effect of exogenously applied dopamine. Furthermore, the inhibitory action of apomorphine on spontaneous activity in the VTA was not potentiated by cocaine. These observations suggest that in low concentrations, cocaine can act as a dopamine reuptake inhibitor in the VTA, and that the resultant increase in extracellular dopamine acts upon dopamine autoreceptors to inhibit cellular activity.

Rat locus coeruleus-hippocampus double in oculo brain grafts were studied with high-speed electrochemical techniques to characterize stimulus-evoked overflow of norepinephrine. Local pressure ejections of KC1 into the locus coeruleus (LC) portion of the double graft elicited electrochemical signals in the hippocampal portion that had NE-like reduction/oxidation current ratios. In contrast, electrical stimulation of the LC part of the double graft did not lead to consistently detectable signals in the hippocampal portion of the transplant. These data demonstrate that LC graft neurons are able to release NE in target tissues when stimulated in an appropriate manner, and that the time course and magnitude of the overflow of NE can be detected with sensitive high-speed electrochemical recording techniques.

The ventral tegmental area (VT A) is a brain region rich in dopamine-containing neurons. Since most agents which act as substrates for self-administration increase dopaminergic outflow in the mesolimbic or mesocortical areas, the VT A slice preparation may be useful for identifying drugs with potential for abuse. While ethanol (EtOH) is a drug of abuse which has been widely studied, the properties of ethanol which contribute to its abuse potential are not known. We have developed a brain slice preparation of the VT A in order to study the action of EtOH on putative dopamine neurons. Concentrations of EtOH from 20 to 320 mM produce a dose-dependent excitation of the dopamine-type neurons of the VTA. About 89% of neurons which have electrophysiological characteristics established for presumed dopamine-containing neurons were excited by ethanol in the pharmacologically relevant concentration range. This excitation persists in low calcium, high magnesium medium, which suggests a direct excitatory action of EtOH on dopamine-type cells in the VT A slice.

Excitatory glutamatergic synapses in the hippocampal CA1 region of rats are potently inhibited by purines, including adenosine, ATP, and ATP analogs. Adenosine A(1) receptors are known to mediate at least part of the response to adenine nucleotides, either because adenine nucleotides activate A(1) receptors directly, or activate them secondarily upon the nucleotides' conversion to adenosine. In the present studies, the inhibitory effects of adenosine, ATP, the purportedly stable ATP analog adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), and cyclic AMP were examined in mice with a null mutation in the adenosine A(1) receptor gene. ATPgammaS displaced the binding of A(1)-selective ligands to intact brain sections and brain homogenates from adenosine A(1) receptor wild-type animals. In homogenates, but not in intact brain sections, this displacement was abolished by adenosine deaminase. In hippocampal slices from wild-type mice, purines abolished synaptic responses, but slices from mice lacking functional A(1) receptors showed no synaptic modulation by adenosine, ATP, cAMP, or ATPgammaS. In slices from heterozygous mice the dose-response curve for both adenosine and ATP was shifted to the right. In all cases, inhibition of synaptic responses by purines could be blocked by prior treatment with the competitive adenosine A(1) receptor antagonist 8-cyclopentyltheophylline. Taken together, these results show that even supposedly stable adenine nucleotides are rapidly converted to adenosine at sites close to the A(1) receptor, and that inhibition of synaptic transmission by purine nucleotides is mediated exclusively by A(1) receptors.

Brief exposure to conditions that generate free radicals inhibits synaptic transmission in hippocampal slices, most likely via a presynaptic mechanism. Because other physiologically stressful conditions that generate free radicals, such as hypoxia or ischemia, stimulate the release of adenosine from brain slices, we determined whether increases in extracellular adenosine mediate the presynaptic inhibition of excitatory transmission induced by peroxide treatment. Simultaneous addition of hydrogen peroxide (0.01%) and ferrous sulfate (100 μM) resulted in a >80% decrease in synaptic potentials recorded in the CA1 region of hippocampal slices of adult male rats. Treatment with theophylline (200 μM), a non-selective adenosine receptor antagonist, or 8-cyclopentyl-1, 3-dipropylxanthine (100 nM), a selective adenosine A 1 receptor antagonist, prior to and during hydrogen peroxide superfusion prevented the inhibition. These results demonstrate that acute exposure to hydrogen peroxide induces an adenosine-mediated decrease in synaptic transmission in hippocampal slices.

This unit describes general techniques that are useful for recording electrophysiological responses that are mediated via the activation of G-protein coupled receptors (GPCRs). It includes a brief description of preparations, but focuses primarily on experiments using hippocampal brain slice preparations. Techniques for the preparation of brain slices, electrodes, filling solutions, and the recording protocols that are suitable for assessing the activity of GPCRs using electrophysiological techniques are summarized, and various protocols for the activation of these receptors are discussed.

The CNS abundantly expresses P2X receptor channels for ATP; of these the most widespread in the brain is the P2X(4) channel. We show that ivermectin (IVM) is a specific positive allosteric effector of heterologously expressed P2X(4) and possibly of heteromeric P2X(4)/P2X(6) channels, but not of P2X(2), P2X(3), P2X(2)/P2X(3,) or P2X(7) channels. In the submicromolar range (EC(50,) approximately 250 nM) the action of IVM was rapid and reversible, resulting in increased amplitude and slowed deactivation of P2X(4) channel currents evoked by ATP. IVM also markedly increased the potency of ATP and that of the normally low-potency agonist alpha, beta-methylene-ATP in a use- and voltage-independent manner without changing the ion selectivity of P2X(4) channels. Therefore, IVM evokes a potent pharmacological gain-of-function phenotype that is specific for P2X(4) channels. We also tested whether IVM could modulate endogenously expressed P2X channels in the adult trigeminal mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM produced no significant effect on the fast ATP-evoked inward currents in either type of neuron, despite the fact that IVM modulated P2X(4) channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric P2X(4) channels are not the primary subtype of P2X receptor in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons.

Many studies have demonstrated that intoxicating concentrations of ethanol (10-100 mM) can selectively inhibit the component of glutamatergic synaptic transmission mediated by N-methyl-D-aspartate (NMDA) receptors while having little or no effect on excitatory synaptic transmission mediated by non-NMDA receptors [i.e., alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and/or kainate (KA) receptors]. However, until the recent development of highly selective AMPA receptor antagonists, it was not possible to assess the relative contribution of AMPA and KA receptors to non-NMDA receptor-mediated synaptic transmission or to determine whether these glutamate receptor subtypes differed in their sensitivity to ethanol. In the present experiments, we used the highly selective AMPA receptor antagonist LY 303070 to pharmacologically isolate KA receptor-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal CA3 pyramidal neurons and tested their sensitivity to ethanol. Concentrations of ethanol as low as 20 mM significantly and reversibly depressed KA EPSCs. Ethanol also inhibited KA currents evoked by direct pressure application of KA in the presence of LY 303070, suggesting that this inhibition was mediated by a postsynaptic action. In contrast, ethanol had no effect on AMPA EPSCs in these cells, even at the highest concentration tested (80 mM). Ethanol significantly inhibited NMDA EPSCs in these neurons, but these responses were less sensitive to ethanol than KA EPSCs. These results suggest that in addition to its well-described depressant effect on NMDA receptor-mediated synaptic transmission, ethanol has an even greater inhibitory effect on glutamatergic synaptic transmission mediated by KA receptors in rat hippocampal CA3 pyramidal neurons.

Primary gustatory afferents from the oropharynx of the goldfish, Carassius auratus, terminate in the vagal lobe, a laminated structure in the dorsal medulla comparable to the gustatory portion of the nucleus of the solitary tract in mammals. We utilized an in vitro brain slice preparation to test the role of different ionotropic glutamate receptor subtypes in synaptic transmission of gustatory information by recording changes in field potentials after application of various glutamate receptor antagonists. Electrical stimulation of the vagus nerve (NX) evokes two short-latency postsynaptic field potentials from sensory layers of the vagal lobe. 6,7-Dinitroquinoxaline-2,3-dione and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, two non-N-methyl-D-aspartate (NMDA) ionotropic receptor antagonists, blocked these short-latency potentials. Slower potentials that were revealed under Mg 2+ -free conditions, were abolished by the NMDA receptor antagonist, D(-)-2-amino-5-phosphonovaleric acid (APV). Repetitive stimulation produced short-term facilitation, which was attenuated by application of APV. These results indicate that the synaptic responses in the vagal lobe produced by stimulation of the gustatory roots of the NX involve both NMDA and non-NMDA receptors. An NMDA receptor-mediated facilitation may serve to amplify incoming bursts of primary afferent activity.

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°C) markedly inhibits excitatory synaptic transmission. This effect was reversible on cooling, readily repeatable, and was blocked by A1receptor antagonists and by adenosine deaminase, suggesting that it was mediated by increased activation of presynaptic adenosine A1receptors 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°C). The increased activity at A1receptors 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.

The study of the actions of ethanol on the brain has generated a wide range of hypotheses concerning the mechanisms by which it alters neuronal activity. These include relatively non-specific actions (e.g., increases in membrane fluidity), that might ultimately alter the function of proteins embedded in the lipid bilayer. However more recent studies have focused on specific interactions between ethanol with membrane proteins, such as neurotransmitter receptors and voltage-gated ion channels. It has been hypothesized that the interaction is between ethanol and a hydrophobic pocket in the protein molecule (Figure 1). The enhancement or antagonism of the function of such membrane proteins has been posited to underlie the alterations in neural activity that ultimately result in the intoxicating effects of ethanol.

Publisher Summary This chapter presents the effects of a selective A 3 agonist, 2 - chloro - N6 - (3 - iodobenzyl)-adenosine - 5' - N - methyluronamide (Cl-IB-MECA), are characterized on hippocampal CA 1 pyramidal neurons. The chapter examines the possibility that activation of A 3 receptors could lead to activation of protein kinase A (PKA), protein kinase C (PKC), or calcium/calmodulin dependent protein kinase II (CaM-K), which might then induce significant changes in the activity of hippocampal pyramidal neurons. CA1 pyramidal neurons were recorded in the whole cell mode using patch electrodes, and the effects of adenosine agonists were determined on the holding current, and on the amplitude of the slow outward calcium-activated K + currents activated following a depolarizing step in the command voltage Z (I AHP ). The result discussed in the chapter suggest that either A 3 receptors are not present on the cell bodies of hippocampal CA1 pyramidal neurons; that they are present but not linked to PKA, PKC, or CaM-K activation; or that they only activate these kinases in cell compartments that are not accessible to the ion channels that underlie the AHP response.

The mechanisms that regulate adenosine release in the brain are not well understood. The present study investigated the hypothesis that individual neurons can generate and release sufficient adenosine to regulate their synaptic inputs. We utilized the whole-cell recording technique to apply enzyme inhibitors and nucleotides directly into the cytoplasm of single rat hippocampal CA1 pyramidal neurons. Cytoplasmic delivery of adenosine induced the release of sufficient adenosine to inhibit excitatory synaptic inputs. However, intracellular delivery of nucleotides and enzyme inhibitors failed to increase adenosine receptor-mediated inhibition. These data suggest that while pyramidal neurons in the hippocampus are capable of releasing large amounts of adenosine into the extracellular space, they do not readily form adenosine from endogenous sources.

Local application of GABA to rat cerebral cortical neurons in brain slices elicited biphasic responses mediated via GABAA receptors. The fast component of the response, which was most apparent with somatic application of GABA, was hyperpolarizing at the normal resting membrane potential (GABAh response). The slower component could be elicited by GABA application to nearly all regions of the cell, and was depolarizing at the resting membrane potential (GABAd response). The reversal potential of evoked IPSCs recorded with whole-cell patch electrodes (-68 mV) was comparable to the reversal potential of the GABAh response (-69 mV), and was significantly different from the reversal potential of the GABAd response (-56 mV). The GABAd response was more sensitive to enhancement by pentobarbital and more readily antagonized by both bicuculline and picrotoxin than the GABAh response. Recording in bicarbonate-free buffer changed the reversal potential of the GABAd response significantly, but had no effect on the GABAh response. In contrast, superfusion with ethanol significantly enhanced the GABAh response, while having no effect on the GABAd component. Although a localized collapse of the Cl- gradient, which has been proposed to underlie the GABAd response, could explain the greater sensitivity of the GABAd response to pentobarbital and the GABAA antagonists, this could not account for the greater sensitivity of the GABAh response to ethanol. Differences in GABAA receptor subunit composition may result in the expression of dendritic and somatic GABAA receptors that have different kinetics, reversal potentials, and sensitivity to pharmacological agents, including ethanol.

The carboxyl terminus of heterotrimeric G protein alpha subunits plays an important role in receptor interaction. We demonstrate that peptides corresponding to the last 11 residues of Galphai1/2 or Galphao1 impair agonist binding to A1 adenosine receptors, whereas Galphas or Galphat peptides have no effect. Previously, by using a combinatorial library we identified a series of Galphat peptide analogs that bind rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). Native Galphai1/2 peptide as well as several analogs were tested for their ability to modulate agonist binding or antagonist-agonist competition using cells overexpressing human A1 adenosine receptors. Three peptide analogs decreased the Ki, suggesting that they disrupt the high affinity receptor-G protein interaction and stabilize an intermediate affinity state. To study the ability of the peptides to compete with endogenous Galphai proteins and block signal transduction in a native setting, we measured activation of G protein-coupled K+ channels through A1 adenosine or gamma-aminobutyric acid, type B, receptors in hippocampal CA1 pyramidal neurons. Native Galphai1/2, peptide, and certain analog peptides inhibited receptor-mediated K+ channel gating, dependent on which receptor was activated. This differential perturbation of receptor-G protein interaction suggests that receptors that act on the same G protein can be selectively disrupted.

The effects of acetylcholine on both pyramidal neurons and interneurons in the area CA1 of the rat hippocampus were examined, using intracellular recording techniques in anin vitroslice preparation. In current-clamp mode, fast local application of acetylcholine (ACh) to the soma of inhibitory interneurons in stratum radiatum resulted in depolarization and rapid firing of action potentials. Under voltage-clamp, ACh produced fast, rapidly desensitizing inward currents that were insensitive to atropine but that were blocked by nanomolar concentrations of the nicotinic α7 receptor-selective antagonists α-bungarotoxin (αBgTx) and methyllycaconitine. Nicotinic receptor antagonists that are not selective for α7-containing receptors had little (mecamylamine) or no effect (dihydro-β-erythroidine) on the ACh-induced currents. Glutamate receptor antagonists had no effect on the ACh-evoked response, indicating that the current was not mediated by presynaptic facilitation of glutamate release. However, the current could be desensitized almost completely by bath superfusion with 100 nmnicotine. In contrast to those actions on interneurons, application of ACh to the soma of CA1 pyramidal cells did not produce a detectable current. Radioligand-binding experiments with [125I]-αBgTx demonstrated that stratum radiatum interneurons express α7-containing nAChRs, andin situhybridization revealed significant amounts of α7 mRNA. CA1 pyramidal cells did not show specific binding of [125I]-αBgTx and only low levels of α7 mRNA. These results suggest that, in addition to their proposed presynaptic role in modulating transmitter release, α7-containing nAChRs also may play a postsynaptic role in the excitation of hippocampal interneurons. By desensitizing these receptors, nicotine may disrupt this action and indirectly excite pyramidal neurons by reducing GABAergic inhibition.

There are multiple mechanisms by which adenine nucleotides can be released into the extracellular space in brain. Adenine nucleotides are converted extracellularly to adenosine, which then acts on adenosine receptors to elicit physiological responses, but the rate at which this conversion takes place is unknown. In the present experiments, adenine nucleotides were applied to individual hippocampal neurons, and the subsequent activation of a postsynaptic K+conductance by adenosine A1receptors was used to determine the rate of adenosine formation. None of the adenine nucleotides tested (cAMP, AMP, ADP, and ATP) activated A1receptors directly at the concentrations tested (≤200 μm). AMP, ADP, and ATP were all rapidly converted to adenosine, with aT1/2for ATP conversion to adenosine of ∼200 msec, and the last step in this pathway (transformation of AMP to adenosine by 5′-nucleotidase) seems to be the rate-limiting step. As we have reported previously, cAMP is converted to adenosine as well, but on a much slower time scale than any of the other nucleotides tested. These experiments demonstrate that fast, localized release of AMP, ADP, or ATP can result in a transient activation of adenosine receptors but that this is unlikely to occur with cAMP. The existence of a highly active ecto-nucleotidase pathway in brain provides a mechanism for the rapid generation of adenosine after the release of adenine nucleotides into the extracellular space.

Publisher Summary Adenosine exerts physiological effects on the nervous system through interactions with cell surface receptors. In the nervous system adenosine has been shown to alter levels of cyclic adenosine 3', 5’-monophosphate (CAMP), inhibit synaptic activity and neuronal excitability, and modulate cerebral blood flow, and these effects have led to numerous proposed functional roles for adenosine. Adenosine can act as a “retaliatory metabolite” by which cells communicate their energy status to the surrounding tissues. Adenosine has been shown to be released during cerebral ischemia, and it increases cerebral blood, inhibits synaptic activity, and decreases neuronal excitability. Thus adenosine acts as a neuroprotectant, preventing damage from ischemia and excitotoxicity. Adenosine receptors are located throughout the nervous system and extracellular adenosine can effectively modulate neuronal activity. This chapter summarizes what is known about how adenosine exerts it effects on neuronal activity, how cells form adenosine and regulate adenosine concentrations, and how cells release adenosine into the extracellular space where it can exert its modulator actions. By integrating these different areas of research, it is hoped that a more complete picture of how the brain uses adenosine as a modulator of synaptic activity will be formed. The chapter discusses the various adenosine receptors, adenosine formation by S-adenosylhomocysteine hydrolase, adenosine formation metabolism of lntracellular nucleotides, degradation of adenosine, adenosine levels in the brain, extracellular degradation of nucleotides, direct release of adenosine into the extracellular space, and localized adenosine release and synaptic modulation. The experimental evidence has made it clear that adenosine serves multiple functions throughout the nervous system. Studies on the actions of exogenous adenosine have conclusively shown that adenosine can act as a potent and effective modulator of synaptic activity and neuronal function. The purpose of the chapter to summarize what is known about how cells synthesize, release, and regulate adenosine.

Adenosine is a potent inhibitory modulator of synaptic transmission in the CNS, but its role in normal physiological function is unclear. In the present experiments, we have found electrophysiological evidence for activity-dependent release of adenosine from hippocampal slices evoked by physiologically relevant stimulation, and have demonstrated that this adenosine modifies synaptic activity in this brain region. When two independent excitatory pathways to the CA1 pyramidal neurons are used to evoke field EPSP responses, prior activation of one pathway will inhibit the EPSP evoked via the other input. This inhibition can be antagonized by the nonselective adenosine receptor antagonist theophylline, and by the selective A1 receptor antagonist 8- cyclopentyltheophylline, suggesting that the inhibitory response is due to the release of endogenous adenosine that activates presynaptic release-modulating A1 receptors. This inhibition can be observed following a single stimulus to the conditioning pathway, although it is more pronounced when a train of conditioning pulses is used, and is maximal following a train of 16–32 stimuli (at 100 Hz). When a train of four conditioning pulses is used, the inhibition appears with a latency of approximately 50 msec, peaks approximately 200–250 msec following the conditioning train, and recovers to baseline between 1 and 2 sec. Further evidence that this inhibition of excitatory transmission is mediated via adenosine is provided by the observation that superfusion with dipyridamole (an adenosine uptake inhibitor), and the adenosine deaminase inhibitor erythro-(2-hydroxy-3-nonyl)adenine, enhanced both the duration and amplitude of the inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand binding studies have demonstrated that bivalent cations can alter interactions between purinergic agonists and adenosine receptors in brain membranes. In the present study, we have investigated whether a similar interaction can be demonstrated in terms of a functional response to adenosine, which is the inhibition of synaptic transmission in the CA1 region of the rat hippocampus mediated via presynaptic A1 receptors. Our data suggest that alterations in cation concentrations do not significantly affect the sensitivity of these adenosine receptors, as long as changes are made in such a way as to leave presynaptic Ca2+ entry unaffected. The experimental results do not support the conclusion that there is a specific effect of either Ca2+ or Mg2+ on adenosine receptor sensitivity, such as has been described for agonist interactions with adenosine A1 receptors. We conclude that the effects of bivalent cations observed in ligand binding studies probably reflect an effect at an intracellular site, either on the receptor itself, or perhaps on associated GTP binding proteins.

Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. The experiments reported here demonstrate that cocaine blocks the induction of LTP at the excitatory synapses in the CA1 region of the hippocampus, but does not appear to do so by blocking NMDA receptors or channels. Once LTP had been established, however, cocaine had no effect on the potentiated response. Cocaine was also able to block LTP initiated by superfusing slices with 25 mM TEA. The ability to block LTP was shared by the local anesthetics lidocaine and procaine, but not by tetrodotoxin, suggesting that the blockade of sodium channels alone did not disrupt LTP. Biochemical experiments demonstrated that cocaine can inhibit phosphorylation of purified Synapsin I by Ca2+/calmodulin-dependent protein kinase II. This effect, presumably mediated by effects on calmodulin, is a previously unreported action of cocaine, and suggests that cocaine at high dose levels might disrupt types of learning that are mediated by an LTP-like mechanism.