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Repetitive stimulation (100 pulses per second for 1 second) of the Schafer collateral-commissural system of the rat hippocampus induces long-term potentiation of synaptic strength and produces significant changes in the subsequent endogenous phosphorylation of a 40,000-dalton protein from synaptic plasma membranes. This effect is not observed after stimulation in calcium-deficient media or after simulation at the rate of one pulse per second for 100 seconds. These findings provide evidence that repetitive synaptic activation can alter the phosphorylation machinery of the synaptic region and suggest a biochemical process which may be involved in the production of neuronal plasticity.

Long sleep (LS) and short sleep (SS) mice have a differential sensitivity to the behavioral actions of an adenosine agonist, R-phenylisopropyl-adenosine (PIA) that parallels their differential sensitivity to the soporific effects of ethanol. In addition to being more sensitive to the sedative effects of PIA, LS mice also show a greater excitatory response to an adenosine antagonist, theophylline (1,3-dimethylxanthine). The brain concentrations of both PIA and theophylline following drug administration do not differ in LS and SS mice, suggesting that the central nervous system of the LS mouse is more sensitive to both adenosine receptor agonists and antagonists. However, LS and SS mice made tolerant to ethanol did not show cross-tolerance to PIA. These results suggest that genetic selection for ethanol sensitivity has resulted in a parallel CNS sensitivity to purinergic drugs, but that acute alterations in sensitivity due to the development of ethanol tolerance do not involve changes in purinergic systems.

In this chapter, we will review studies of electrophysiological actions of ethanol on central neurons which may be correlated with the acute soporific effects of this drug. Since there have been several excellent reviews surveying the actions of ethanol on neuronal excitability at many levels of the neuraxis (Berry and Pentreath, 1980; Deitrich and Spuhler, 1984; Himwich and Callison, 1972; Klemm, 1979; Seiger et al., 1983; Siggins and Bloom, 1980), we should like to focus this review on those aspects of neuronal sensitivity to the effects of ethanol that may arise primarily as a result of genetic selection for acute sensitivity to ethanol-induced ataxia.

Long-term potentiation (LTP), a lasting increase in synaptic efficacy seen following high-frequency stimulation, has been extensively studied as a mnemonic model. However, a major conceptual difficulty in relating LTP to an endogenous mechanism for memory formation is that the parameters commonly used for its induction are well beyond the normal physiological firing range of hippocampal neurons: usually 50–400 stimuli, delivered at frequencies of 100–400 Hz, are employed (Teyler and Discenna, 1984). While hippocampal pyramidal cells are known to discharge in “complex-spikes”, consisting of several action potentials at frequencies greater that 100 Hz, groups of 3 or 4 action potentials are most commonly seen (Suzuki and Smith, 1985); continuous high frequency discharge is only observed under experimentally-induced or pathological conditions. We considered the possibility that the threshold for the induction of LTP might be reduced if the pattern of stimulation more accurately reflected normal neuronal firing.

Norepinephrine (NE) has been shown to have a biphasic effect on evoked potentials in the CA1 region of the hippocampus of the rat in vitro, with a beta receptor mediating an increase and an alpha receptor eliciting a decrease in the amplitude of the population spike. The purpose of this study was to use selective alpha-adrenergic agonists and antagonists to determine the subtype of receptor mediating the depressant response of NE. The present investigations demonstrated that the selective alpha1 agonist, phenylephrine (2–50 μ M) elicited a dose-dependent depression of the amplitude of the population spike. Clonidine, a relatively selective alpha2-agonist, also depressed the amplitude of the population spike, but only at concentrations (10 μ M) that were inconsistent with a selective action upon alpha2-receptors. Another alpha2-agonist, alpha-methylnorepinephrine (100–400 nM) did not depress the amplitude of the population spike. The depressant effect of NE was antagonized by the nonselective alpha antagonist, phentolamine (0.5–50 μ M) and the alpha1-selective antagonist, prazosin (1 μ M), but not by the alpha2-selective antagonist, idazoxan (1–10 μ M). Phentolamine and prazosin antagonized the response to phenylephrine but not to clonidine. The depressant effect of NE was not antagonized by the antagonist of serotonin and dopamine, spiperone (100 nM); conversely, the effect of 8-hydroxy-2-(di-n-propylamine) tetralin (50 μ M), a 5-HTA receptor-selective agonist, which also depresses the amplitude of the population spike, was not antagonized by phentolamine (5 μ M). These data indicate that, in the in vitro slice preparation, NE acts upon an alpha1-adrenergic receptor to depress the amplitude of the population spike, and not an alpha2-adrenergic receptor, a 5-HT1A receptor or a dopamine receptor. The data also support the conclusion that serotonin acts independently to reduce the response of the hippocampal population spike.

Biochemical studies have shown that the non-barbiturate anesthetic etomidate can interact in a stereoselective, barbiturate-like fashion with the GABA/benzodiazepine receptor complex, enhancing both benzodiazepine and GABA binding, but its electrophysiological effects upon the mammalian CNS are largely unknown. The present investigations were designed to characterize the electrophysiological effects of etomidate on the recurrent GABAergic inhibitory pathway in the CA1 region of the rat in vitro hippocampal slice and to compare the actions of etomidate to those of pentobarbital. Electrical stimulation of the alveus elicited a biphasic hyperpolarizing response, consisting of an initial bicuculline-sensitive GABAergic IPSP. This was followed by a second component, termed the late hyperpolarizing potential (LHP), which is thought to reflect an increase in potassium conductance. Both pentobarbital (100 microM) and (+)-etomidate (10 microM) markedly increased the duration of the initial GABA-mediated IPSP, and frequently increased its amplitude as well. However, no significant effects of either of these drugs were observed on the LHP. Together with previous biochemical findings, our data suggest that the depressant effects of etomidate and barbiturates on the nervous system may reflect a common action upon a stereoselective receptor site intimately associated with bicuculline-sensitive GABA receptors and the chloride ion channel.

Electrophysiological 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.

Publisher Summary This chapter discusses the physiological role of adenosine in the central nervous system (CNS). In particular, the chapter's 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.

Superfusion of Purkinje neurons in the in vitro rat cerebellar slice with norepinephrine caused increases and decreases of spontaneous Purkinje cell firing. Excitations were evoked by low concentrations of norepinephrine (0.5-10 microM) and by the beta receptor agonist isoproterenol (0.1-5 microM). These excitations were reduced by timolol (1-2 microM), a beta receptor antagonist. Perfusion with higher concentrations of norepinephrine (greater than 16 microM), caused a depression of Purkinje neuron spontaneous activity. This inhibitory response was blocked by the alpha receptor antagonist phentolamine. The alpha 1 selective agonist phenylephrine had no effect on spontaneous activity at concentrations up to 100 microM, but the alpha 2 selective agonist clonidine (1-50 microM) elicited decreases in firing rate. These responses appeared to be due to a direct action on Purkinje cells, because neither the excitation nor the depression of Purkinje neuron activity elicited by norepinephrine was substantially altered when tested in a medium which substantially blocked synaptic transmission within the slice. Under these in vitro conditions, norepinephrine appears to increase the firing rate of Purkinje neurons via an interaction with beta adrenergic receptors, while norepinephrine induced depressions may be linked to alpha adrenergic receptor interactions; both receptors appear to be located directly on the Purkinje neurons.

The electrophysiological effects of phencyclidine were examined in the CA region of slice preparations of the hippocampus of the rat. The only direct effects of phencyclidine on monosynaptic transmission in the Schaffer collateral/commissural input to the CA region were local anesthetic-like actions, that were observed at concentration greater than or equal to 50 microM, and were characterized by decreases in the amplitude of both orthodromically- and antidromically-evoked potentials. However, phencyclidine, at concentrations greater than 0.5 microM, increased the apparent sensitivity of slices to perfusion with norepinephrine (NE), such that a previously subthreshold concentration of NE significantly increased the magnitude of the population spike response through an action at beta-adrenergic receptors. This action of phencyclidine appears to reflect its ability to block the high affinity uptake of NE into nerve terminals in the slice preparation. Because these responses were observed at concentrations of phencyclidine which are found in the brains of abusers of phencyclidine, they may play a significant role in the overall neuropharmacological response to phencyclidine.

The effect of pertussis toxin pretreatment on electrophysiological responses to selective delta- and mu-opioid receptor agonists was examined in rat hippocampal brain slices. The evoked population spike response in the CA1 region following activation of the Schaffer collateral and commissural afferents was increased following perfusion with the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), and with the mu-selective agonist [D-Ala2,NMe-Phe4,Gly(O)5ol]enkephalin (DAGO). Both effects were significantly reduced or abolished in brain slices obtained from animals that had been pretreated with pertussis toxin 2-3 days earlier. These findings suggest that the excitatory responses to opioid agonists in hippocampus are the result of interactions with receptors that are coupled via pertussis toxin sensitive GTP-binding proteins to their respective effector mechanisms.

The recently discovered profound differential sensitivity of cerebellar Purkinje (P) cells in long-sleep (LS) verus short-sleep (SS) mice to the depressant effects of locally applied ethanol was extended in this study. First, the sensitivity of Purkinje neurons from HS mice (an outbred stock of mice from which the LS and SS lines were derived), was found to be almost exactly intermediate between the values for the long-sleep and short-sleep animals. Second, no differential sensitivity in long-sleep versus short-sleep hippocampal pyramidal neurons was observed. This was true using both spontaneous and evoked activity. Third, no differential sensitivity of P cells was seen in long- versus short-sleep mice with local application of halothane. Taken together with previous reports, these data strongly suggest that whatever genetically determined central nervous alterations result in the differential soporific effects of ethanol in the two (LS and SS) mouse lines, such alterations are brain region- and depressant drug-specific rather than generalized.

Behavioral responses to an adenosine receptor agonist and antagonist were examined in mice genetically selected for differential sensitivity to the soporific effects of ethanol. Both ethanol and the adenosine receptor agonist L-phenylisopropyladenosine had greater sedative and hypothermic effects in ethanol-sensitive "long-sleep" mice than in ethanol-insensitive "short-sleep" mice. Long-sleep mice were also more sensitive to the excitatory behavioral effects of theophylline, an adenosine receptor antagonist. These data suggest that adenosine may be an endogenous mediator of responses to ethanol.

Adenosine has profound depressant effects upon the electrophysiological activity of the brain, but the adenosine receptor subtypes which mediate these responses are uncertain. In order to resolve this question, we have characterized the effects of two adenosine analogs which differ in their relative potencies at adenosine A1 and A2 receptors. The effects of these adenosine analogs were examined on spontaneous firing rate of Purkinje neurons in the rat cerebellum in situ, in cerebellar brain slices in vitro, and on synaptic transmission in the rat hippocampus in vitro. Although the A2 agonist appeared to be more potent with local drug application techniques in situ, our in vitro results suggest that the A1 receptor subtype is involved in the electrophysiological actions of these drugs in both rat cerebellum and hippocampus. Furthermore, these data indicate that the physical properties of some adenosine analogs may reduce apparent drug potencies when they are studied with local application techniques.

The effect of varying calcium and magnesium concentrations was observed on the development of long-term potentiation (LTP) in the CA1 region of the rat hippocampus maintained in vitro. Treatments which would be expected to lower the depolarization-induced influx of calcium into neuronal elements (i.e. lowering Ca2+ or raising Mg2+ in the perfusion medium) selectively antagonized LTP, while synaptic transmission, paired-pulse and frequency facilitation, as well as short-term (or post-tetanic) potentiation were all maintained. This suggests that LTP is unrelated to these short-term forms of plasticity, but is either a calcium-dependent process itself, or is dependent upon some other calcium-related phenomenon (such as transmitter release).

The effects of cocaine on electrophysiological activity were examined in the CA1 region of the in vitro hippocampal slice. Low concentrations of cocaine (0.25-2.5 microM) had no direct effect on evoked potentials, but potentiated responses to threshold concentrations of norepinephrine (NE; 0.5 microM); this effect was also seen with other inhibitors of catecholamine uptake such as desipramine. The ability of low doses of cocaine to augment the changes in population spike amplitude elicited by NE appeared to be related to the ability of cocaine to block the uptake of NE into the noradrenergic nerve terminals in the rat hippocampus. Higher concentrations of cocaine (5-200 microM) elicited responses consistent with local anesthetic actions on neuronal membranes.

Diazepam (5 x 10(-8) - 10(-6) M) was found to augment recurrent inhibition of pyramidal neuron firing in a dose-dependent manner in rat hippocampal slices. To determine possible loci of this effect, diazepam was locally administered by pressure ejection from a micropipette, while recording action potentials from single inhibitory ("basket") interneurons. Diazepam induced reversible and reliable increases in interneuron firing in response to stimulation of Schaffer collateral and commissural afferents. Taken together with previous electrophysiological reports, these data suggest that benzodiazepines may augment central inhibition by increasing either the excitability of inhibitory interneurons, or by increasing the strength of excitatory afferents to these cells.

Following profound (greater than 90%) depletions of norepinephrine (NE) by the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4), the numbers of beta-adrenergic receptors were significantly increased (20-25%) in rat hippocampal and somatosensory cortical membranes; however, the numbers of alpha 1-adrenergic receptors and the affinities of both types of receptors were unaffected. This selective up-regulation of beta-adrenergic receptors was evident 1 week after DSP4 administration and was maintained for at least 2 more weeks. In electrophysiological experiments in the hippocampal slice preparation, responses to threshold as well as maximal concentrations of isoproterenol were enhanced 150% and 33%, respectively, in the DSP4-lesioned animals. The results demonstrate that nearly complete depletion of brain NE produced by administration of DSP4, like that produced by 6-hydroxydopamine, results in increased numbers of beta- but not alpha-adrenergic receptors, and suggest that the density of the former are regulated by afferent noradrenergic fibers. Furthermore, the functional significance of the increased number of hippocampal beta-adrenergic receptors is directly manifested in a greater electrophysiological responsiveness to an exogenously administered beta-adrenergic receptor agonist.

Although adenosine and its analogs have potent effects on the peripheral and central nervous systems, the actions of systemically administered adenosine analogs on the central nervous system are poorly understood. We have previously shown that adenosine analogs depress hippocampal-evoked responses (fEPSPs) in a brain slice preparation, and in the present study, we observed that local pressure ejection of adenosine or R-phenylisopropyladenosine (R-PIA) also reduced the fEPSP recorded in situ in a theophylline-reversible manner. However, systemic administration of R-PIA in up to 1000 times the behaviorally active dose failed to attenuate the fEPSP amplitude. Similar observations were made with systemic injections of 2-chloroadenosine and N-ethylcarboxamidoadenosine, both of which are active in behavioral studies following intraperitoneal or intracerebroventricular injection. Autoradiography showed systemically injected [3H]PIA did not enter the brain in significant concentrations, and this observation was confirmed using microdialysis brain perfusion. These results suggest that systemically administered adenosine analogs do not affect synaptic neurotransmission in the hippocampus because they fail to reach the appropriate receptors.

In addition to potentially improving the symptoms and the course of certain neurodegenerative diseases, brain tissue grafts may have even greater importance in the study of factors that lead to age-related alterations in the brain. Several strategies for studying this possibility are discussed.