Thomas V. Dunwiddie

The physiological role of adenosine in the central nervous system.

Publisher Summary This chapter discusses the physiological role of adenosine in the central nervous system (CNS). In particular, the chapters attention is directed to the actions of adenosine and related purines in the CNS. One of the major advances in the understanding of purine actions in the central nervous system is the characterization of high affinity specific receptors for purines in brain membranes. The physiological actions of purines, the receptors and biochemical actions that underlie these functional responses and behavioral responses to purinergic drugs are considered. There are three different adenosine receptor sites in brain, which can be distinguished primarily by their effects upon adenylate cyclase, their pharmacological properties, and by their ability to bind labeled analogs of adenosine. One of the best-characterized physiological actions of adenosine is the inhibition of the release of neurotransmitters, an action that has been unequivocally established at many peripheral synapses. If one thing remains clear, it is that purines have a multitude of complex actions at every level, including the biochemical, physiological, and behavioral. There remains little question, but that adenosine or other purines constitute important and rather ubiquitous regulators of neuronalactivity in brain. Despite the evidence that purines play a significant role in neural function, it has remained difficult to define the functional role(s) of purines in brain.

Ethanol increases the firing rate of dopamine neurons of the rat ventral tegmental area in vitro

The ventral tegmental area (VTA) 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 VTA 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 VTA 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 VTA slice.

Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor

Caffeine is believed to act by blocking adenosine A1 and A2A receptors (A1R, A2AR), indicating that some A1 receptors 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.

Ethanol sensitivity of the GABAA receptor expressed in xenopus oocytes requires 8 amino acids contained in the γ2L subunit

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.

Synaptic Potentials Mediated via a-Bungarotoxin-Sensitive Nicotinic Acetylcholine Receptors in Rat Hippocampal Interneurons

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 μm nicotine, 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.

Induction of hippocampal long-term potentiation using physiologically patterned stimulation

Lasting increases in the population spike recorded in area CA1 of hippocampal slices may be evoked by the patterned presentation of as few as 5 stimulus pulses delivered to the commissural/associational afferents. The most effective pattern of stimulus presentation was a single priming pulse followed 170 ms later by 4 pulses at 100 Hz; control trains of 5 pulses at 100 Hz had no significant enduring effect. This pattern-dependent phenomenon, termed primed burst potentiation, is of lesser magnitude than is the long-term potentiation induced by 100 Hz/l s stimulation, but appears to show a similar time course and duration. In addition, the two phenomena are not additive, suggesting that they may share a common mechanism.

Endogenously Released Adenosine Regulates Excitability in the In Vitro Hippocampus

The role of adenosine in regulating epileptiform discharge was studied in the in vitro hippocampal slice preparation. Exogenously applied adenosine appeared to have anticonvulsant properties (EC50∼ 10 μM), in that it slowed or suppressed spontaneous interictal discharges. In addition, the release of adenosine (or related adenine nucleotides) from the slice preparation appeared to exert a tonic inhibitory influence on these types of discharges. This provides further evidence supporting a role for adenosine as an endogenous regulator of the excitability of the central nervous system, particularly in convulsive states.

Noradrenergic responses in rat hippocampus: Evidence for mediation by α and β receptors in the in vitro slice

Summary The effect of perfused norepinephrine (NE) on evoked potentials in CA1 of the in vitro rat hippocampus was examined. Weak and variable effects on population spike amplitude were observed, with lower doses of NE generally producing excitations and higher doses more often producing inhibitions. Clonidine, an α-receptor agonist, produced a dose-dependent inhibition of population spike amplitude; this inhibition was effectively antagonized by the α-antagonist, phentolamine. Isoproterenol (ISO), a β-agonist, produced marked increases in population spike amplitude which could be antagonized by timolol, a β-receptor antagonist. Phentolamine did not antagonize the excitations produced by ISO, and timolol had no effect on the inhibitions seen with clonidine. After pretreatment with either phentolamine or timolol, NE perfusion elicited robust and consistent elevations or reductions in the population spike, respectively. A potent cyclic AMP derivative, 8-p-chlorophenylthio cyclic AMP, produced large increases in population spike amplitude which appeared similar to the responses seen with β-agonists. No changes in field EPSP amplitudes were observed with any of the drugs tested. Taken together, these results suggest that NE may interact with α-adrenergic receptors to decrease pyramidal cell excitability, and with β-adrenergic receptors to increase pyramidal cell excitability; the β-effect may involve cAMP.

Alkylxanthines elevate hippocampal excitability

SummaryThe effects of four xanthines (theophylline, 8-phenyltheophylline, isobutylmethylxanthine (IBMX), and 7-benzyl-IBMX) were studied in the hippocampal slice in vitro. These agents increased the excitability of this preparation with 8-phenyltheophylline being the most potent, 7-benzyl-IBMX the least potent, and theophylline and IBMX having intermediate potencies. A similar rank order was observed in terms of the potencies of these xanthines in antagonizing a) electrophysiological responses to adenosine, and b) adenosine-stimulated cyclic AMP formation. These results indicate that the excitatory actions of xanthines in the in vitro hippocampus can be most easily explained on the basis of their ability to block adenosines actions; the CNS excitatory actions of these drugs in vivo may depend upon a similar mechanism of action.

Adenosine receptors mediating inhibitory electrophysiological responses in rat hippocampus are different from receptors mediating cyclic AMP accumulation

SummaryElectrophysiological and biochemical techniques were used to characterize adenosine receptors in rat hippocampus. The site which mediates the inhibitory action of adenosine on excitatory synaptic transmission and on spontaneous interictal spiking had properties similar to the adenosine A1 receptor. Thus, the relative order of potency for adenosine analogs was l-PIA≥CHA>NECA> 2CA (l-PIA = N6-phenylisopropyladenosine; CHA = N6-cyclohexyl-adenosine; NECA = adenosine 5′-ethylcarboxamide; 2CA = 2-chloroadenosine), with EC50 values for the most potent analogs between 10–30 nM. The effect of the stable adenosine analog, particularly CHA and l-PIA, was slow in onset and very slowly reversible. This is suggested to be due both to a slow dissociation of these compounds from the receptors but particularly to the slow equilibrium between the concentration of the drug in the medium surrounding the slices and the biophase within the slices. Adenosine analogs bound specifically to membrane preparations of the rat hippocampus with the order of potency 3H-CHA≥3H-l-PIA>3H-NECA. Eadie-Hofstee plots of the binding data were curvilinear for each ligand, but only for 3H-l-PIA could the existence of two binding sites with different apparent Kd-values (0.27 and 11.8 nM) be confirmed by curve-fitting. The estimated Kd-values for CHA and NECA were 1.5 and 20 nM, respectively. The adenosine analogs also enhanced 3H-cyclic AMP accumulation in 3H-adenine-labelled hippocampal slices. The rank order of potency of adenosine analogs in increasing cyclic AMP (NECA>2CA>l-PIA>CHA) suggests that this effect is mediated by adenosine A2 receptors. The EC50 values for the accumulation of cyclic AMP were 10–1000 × higher than the EC50 values derived from electrophysiological experiments and the Kd-values from measurements of radioligand binding. Thus, on the basis of absolute as well as rank order potencies of drugs, the adenosine analog-induced electrophysiological responses appear to be related to actions at an A1 receptor site. By contrast, the adenosine receptor-mediated increases in cyclic AMP appears to involve an A2 receptor, the functional role of which is not clear.