LE JOURNAL CANADIEN DES SCIENCES NEUROLOGIQUES

The Role of Cyclic Nucleotides in the CNS

JOHN W. PHILLIS

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CONTENTS A. Introduction 153 (4) Histamine 161 (5) Adenosine 161 B. General Features of Chemical (6) Acetylcholine and Substance P 162 Transmission Between Nerve Cells ...154 (7) Amino Acids 162 I. Electrophysiology of Synaptic (8) Prostaglandins 162 Transmission 154 (1) Fast Synapses 154 E. Nucleotides and Transmission (2) Slow Synapses 155 at Selected Synapses 163 (3) Presynaptic Inhibition 156 I. Sympathetic Ganglia 163 II. Identification of Synaptic Transmitters ...156 II. Central "Aminergic" Transmission 165 (1) Norepinephrine-Mediated Synapses .... 165 C. Metabolism and Functional (a) Cerebellar Purkinje Cells Characteristics of Cyclic and Hippocampal Pyramidal Neurons 165 Nucleotides 157 (b) Cerebral Cortex 165 I. Components of the Cyclic Nucleotide (c) Spinal Motoneurons 166 System (2) Dopamine-Mediated Synapses 166 (1) Adenylate Cyclase 157 (3) Serotonin-Mediated Synapses 167 (2) Guanylate Cyclase 157 (4) Histamine-Mediated Synapses 167 (3) Protein Kinases 157 (4) Phosphodiesterase 158 III. Depressant Actions of Cyclic and Non- (5) Phosphoprotein Phosphatases 158 Cyclic Adenine Nucleotides 168 (1) Cerebellar Purkinje Cells 168 II. Criteria for Acceptance of Cyclic (2) Cerebral Cortical Neurons 170 Nucleotides as Second Messengers 158 (3) Other Regions of the Brain 173 (4) Spinal Cord 173 D. Formation of Cyclic IV. Presynaptic Actions of Cyclic AMP '74 Nucleotides in the CNS 159 V. Acetylcholine and Cyclic GMP 176 I. Preparations used for Studies on VI. Phosphodiesterase Inhibitors 177 Cyclic Nucleotides 159 VII. Prostaglandins 177 (1) Investigations in vivo 159 (2) Brain Slice Preparations 159 F. Amines, Calcium and Na+ , K+ (3) Cells from Neuronal or Glial Cultures 160 —ATPase 178 (4) Cell-free Preparations of Adenylate Cyclase 160 G. Cyclic Nucleotides and Memory 183 II. Neurotransmitters Affecting Cyclic Nucleotide Formation 160 H. Conclusions 184 (1) Norepinephrine 160 (2) Dopamine 161 I. Acknowledgements 185 (3) Serotonin 161 J. References 185

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The Role of Cyclic Nucleotides in the CNS

JOHN W. PHILLIS

SUMMARY: On the basis of the infor­ pense of new difficulties. Prior blockade A. INTRODUCTION mation presented in this review, it is dif­ of the adenosine receptor with agents Cyclic AMP (cAMP, adenosine ficult to reach any firm decision regard­ such as theophylline or adenine xylofura- 3', 5'-monophosphate) was initially ing the role of cyclic AMP (or cyclic noside may also assist in the categoriza­ GMP) in synaptic transmission in the tion of responses to extracellularly ap­ discovered by Sutherland and Rail brain. While it is clear that cyclic nucleo­ plied cyclic AMP as being a result either (1958) as a heat stable factor ac­ tide levels can be altered by the ex­ of activation of the adenosine receptor cumulated in liver homogenates ex­ posure of neural tissues to various neuro­ or of some other mechanism. Ultimately, posed to epinephrine or glucagon transmitters, it would be premature to the development of highly specific in­ which mediated the glycogenolytic claim that these nucleotides are, or are hibitors for adenylate cyclase should action of these hormones on the not, essential to the transmission pro­ provide a firm basis from which to liver. Further investigations have led cess in the pre- or postsynaptic compon­ draw conclusions about the role of cyclic to the concept that cyclic AMP may ents of the synapse. In future experi­ AMP in synaptic transmission. Similar be involved as an intracellular ments with cyclic AMP it will be neces­ considerations apply to the actions of mediator of the actions of other sary to consider more critically whether cyclic GMP and the role of its synthe­ hormones and of various putative the extracellularly applied nucleotide sizing enzyme, guanylale cyclase. merely provides a source of adenosine synaptic transmitters. According to The use of phosphodiesterase in­ this concept, first messengers, the and is thus activating an extracellularly hibitors in studies on cyclic nucleo­ located adenosine receptor, or whether it hormones or transmitters them­ tides must also be approached with cau­ is actually reaching the hypothetical tion. The diverse actions of many of selves, travel from their cells of sites at which it might act as a second these compounds, which include calcium origin and induce the formation of messenger. The application of cyclic mobilization and block of adenosine up­ intracellular cyclic AMP in their AMP by intracellular injection techni­ take, could account for many of the re­ target cells. Cyclic AMP, by activat­ ques should minimize this particular ing an appropriate sequence of en­ problem, although possibly at the ex- sults that have been reported in the lit­ erature. zymes, can evoke the specific re­ sponse of a target cell to the hor­ mone (Sutherland et al., 1968). RESUME: En se basant sur Vinforma­ ment une source d'adenosine et active tion donnee dans cette revue, il est ainsi un receptuer d'adenosine localise Examples of systems in which difficile d'en arriver a une decision extracellulairement, ou si il rejoint en cyclic AMP is thought to function in ferme en ce qui concerne le role de fait les sites hypotheliques qui peuvent a second messenger capacity include VAMP cyclique (ou GMP cyclique) dans agir comme second messager. Lappli­ the liver, where as noted above cyc­ la transmission synaptique dans le cer- cation d AMP cyclique par techniques lic AMP mediates the actions of veau. Tandis qu'il est clair que les d'injection intracellulaire doivent mini- epinephrine and glucagon by mod­ niveaux de nucleotide cyclique peuvent miser ce probleme particulier, quoique ulating carbohydrate metabolism, i'tre modifies par Vexposition des tissus possiblement au coiit de nouvelles diffi- stimulating glycogen breakdown and nerveux a divers neurotransmetteurs, il cultes. Un bloc prealable du recepteur serail premature de pretendre que ces d'adenosine avec des agents comme la promoting the formation of glucose nucleotides sont, ou ne sont pas, essen- theophylline ou I'adenine xylofuranoside from metabolites such as amino tiels au processus de transmission dans peut aussi aider dans la classification acids and lactate. In pancreatic cells, les composantes pre- our post-synap- des reponses a I'AMP cyclique appli- it stimulates the secretion of insulin tiques de la synapse. Dans les experi­ quee extracellulairement, comme etant in response to glucagon and possibly ences futures avec I AMP cyclique, il un re suit at soit d'activation du recepteur to glucose. In fat cells it mediates the sera necessaire de considerer de facon dadenosine ou d'autres mecanismes. actions of hormones such as epine­ plus critique se le nucleotide applique Finalement, le developpement d'inhibi- phrine and glucagon which stimulate extracellulairement procure simple- (Continued on page 185) lipolysis and inhibit lipogenesis. Similar processes involving cyclic From the Department of Physiology, College of AMP are thought to govern other Medicine, University of Saskatchewan, Saskatoon, types of secretory cells. For exam­ Saskatchewan. ple, the actions of many trophic Reprint requests to Prof. J. W. Phillis, Dept. of hormones (ACTH, TSH, FSH) may Physiology, College of Medicine, University of be mediated through cyclic AMP. In Saskatchewan, Saskatoon, Saskatchewan, Canada. S7N 0W0. kidney tubule cells, cAMP stimu-

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lates the reabsorption of water in an increasing awareness of the exis­ cyclic nucleotide function, such as response to antidiuretic hormone; in tence of extracellular adenosine re­ the generation of neuronal memory the myocardium it enhances the rate ceptors on nerve cells. Activation of traces at the single cell level, will be and force of cardiac contraction in these receptors must therefore be introduced. response to epinephrine; and in the considered as a possible complica­ bones it mediates the action of tion when cAMP is administered ex- parathyroid hormone in stimulating tracellularly — the route adopted by B. GENERAL FEATURES OF bone resorption and thus regulates most investigators. CHEMICAL TRANSMISSION the levels of serum calcium. Cyclic The present chapter will examine BETWEEN NERVE CELLS AMP has also been implicated in current knowledge of the role of Before proceeding to a description many of the processes of cell regula­ cyclic nucleotides in synaptic trans­ of the components of the cyclic nuc­ tion in which hormones may not be mission in the central nervous sys­ leotide system in the central nervous directly involved. tem. In particular, the possibility system, it is essential to understand During the course of research on that cyclic nucleotides act as in­ the general processes involved in the cAMP, a second cyclic nucleotide, tracellular mediators of the action of transmission of information between guanosine 3', 5'-monophosphate various neurotransmitters will re­ nerve cells. As the identity of the (cGMP) was discovered (Ashman et ceive a critical examination. To chemicals which convey this infor­ al., 1963). Cyclic GMP is now achieve this objective it will first be mation across the synapses is still thought to act as a second messenger necessary to describe briefly the na­ controversial, it is also necessary to for the actions of various neurotrans­ ture of chemical transmission be­ introduce a summary of the criteria mitters, including acetylcholine. tween nerve cells and to introduce by which these transmitters are ten­ Biomedical research into the the reader to the putative transmitter tatively identified. mediator roles of cyclic nucleotides substances which are thought to has expanded in an explosive fash­ mediate intercellular transmission. I. ELECTROPHYSIOLOGY OF ion in the past two decades and, as The electrophysiology of synaptic SYNAPTIC TRANSMISSION already mentioned, these com­ transmission will be described with Chemically transmitting synapses pounds have been implicated in a special attention devoted to the are divisible into two categories. At plethora of biological processes. A so-called slow synaptic phenomena, what may be called the "classical" particularly intriguing question is which have time courses longer than type of synapse, the presynaptically how can one or two compounds ac­ those traditionally associated with released transmitter acts upon re­ complish such a multitude of roles? chemical transmission. ceptors on the subsynaptic mem­ It must be appreciated from the out­ The third section of this paper brane to cause a rapid increase in set that the detailed mechanisms of discusses in some depth the various membrane permeability to selected hormone-cAMP action have been components of the cyclic nucleotide ions. More recently another type of elucidated for only one system — systems, including enzymatic forma­ chemical synapse has been identified the epinephrine and glucagon- tion, degradation and expression of at which the transmitter alters the induced regulation of glycogen nucleotide action, within the single membrane potential of the post­ breakdown and synthesis. The cell. The traditional criteria for ac­ synaptic cell in the absence of any biological effects of cyclic nuc­ ceptance of cyclic nucleotides as in­ decrease in membrane resistance; leotides generated in response to tracellular mediators of transmitter indeed resistance may even be in­ neurotransmitters are thought to be or hormonal action are then enumer­ creased. At some of these latter the result of activation of a class of ated. types of junction it appears that the transmitter alters the activity of enzymes known as protein kinases, An account of the effects of the metabolic systems in the postsynap­ and the specificity and nature of the putative neurotransmitters on cyclic tic neuron. respofise depend on the substrate nucleotide formation in sympathetic specificity of the particular kinase ganglia and the central nervous sys­ (1) Fast Synapses affected and the accessibility of en­ tem follows with a detailed analysis At the excitatory synapses formed dogenous protein substrates. Cur­ of the electrophysiological, phar­ by dorsal root afferents on spinal rently, however, there is little perti­ macological and cytochemical evi­ cord motoneurons excitatory trans­ nent information regarding the mac- dence favoring an involvement of mission is now considered to have romolecular components which link cyclic nucleotides at certain chemical and possibly electrical these kinases to alterations in catecholamine-mediated synapses. components. The excitatory post­ neuronal excitability. The possibility that other membrane synaptic potential (EPSP) generated Other problems which have ham­ constituents such as Ca++ and by activity in these fibers has an pered the establishment of cyclic Na+ , K+ -ATPase may be involved extremely short time course, which nucleotides as second messengers in the expression of neurotransmit­ is associated with an increase in for the expression of transmitter ac­ ter action, either in conjuction with, membrane conductance and a rever­ tion include the difficulty of estab­ or independently of cyclic nuc­ sal potential more positive than the lishing whether effects occur in the leotides, is explored in some detail. membrane potential (Rail et al., pre- or post-synaptic elements and Finally, other potential aspects of 1967; Jack et al., 1971). The lack of

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clear conductance changes or a re­ paralleled by changes in the reversal erated by a decrease in the resting versal level for the EPSP's recorded potential for the IPSP, suggesting potassium conductance of the mem­ from some motoneurones has been that alterations in chloride conduc­ brane. Metabolic inhibitors such as ascribed to a component of electrical tance are of major significance in the 2,4-dinitrophenol and sodium azide transmission. The currents which generation of the IPSP. Another ex­ appear to have a selective inhibitory generate the EPSP have been meas­ ample of a chloride-dependent IPSP action on the slow EPSP (Kobayashi ured directly by voltage clamp­ has been established in cat trochlear and Libet, 1968), suggesting that ac­ ing monosynaptically activated motoneurons, where the IPSP has a tivation of metabolic pathways in the motoneurons (Araki and Terzuolo, reversal potential that is more nega­ postsynaptic neuron may be in­ 1962). The current has an initial in­ tive than the potassium-dependent volved in its generation. tense phase which rises to a peak in action potential after hyperpolariza­ The behavior of the slow IPSP of about 0.7 msec and a total duration tion (Llinas and Baker, 1972). sympathetic ganglion cells is not that of about 2.5 msec. Current flow is These IPSP's are reversed by in­ which would be expected of a poten­ possible because of the increase in tracellular chloride injection, imply­ tial generated by increased mem­ postsynaptic membrane permeabil­ ing that chloride ion carries most of brane permeability to certain ions. ity to sodium and potassium ions the IPSP current and that intracel­ that accompanies activation by the Membrane resistance may either be lular chloride is actively maintained unchanged (rabbit) (Kobayashi and excitatory transmitter of its recep­ at a lower concentration than would tors. Libet, 1968) or increased (frog) be expected from passive chloride (Weight and Padjen, 1973a). During The inhibitory synaptic action of distribution. depolarization the slow IPSP is la afferents on antagonist depressed, reaching zero at about 20 motoneurons in the spinal cord is (2) Slow Synapses The most fully documented ex­ mV depolarization. With moderate revealed as a brief hyperpolarization hyperpolarization the IPSP is in­ of the membrane. '?he inhibitory amples of synapses at which trans­ mission occurs in the absence of a creased in size (Nishi and Koketsu, postsynaptic potential (IPSP) is ap­ 1968; Libet, 1970). proximately a mirror image of the la decrease in membrane resistance are EPSP and is generated by an out­ found in the sympathetic ganglia of Libet and his colleagues (Libet ward current across the subsynaptic mammals and amphibians. The slow and Tosaka, 1968; Libet and Ow- membrane. The inhibitory current, EPSP generated at muscarinic man, 1974) have proposed that dopa­ as measured by voltage clamp tech­ cholinergic receptors on the ganglion mine is the specific synaptic trans­ niques (Araki and Terzuolo, 1962) cell membrane occurs either with no mitter responsible for generation of has a rapid time course, reaching a change in resistance (rabbit, frog) the slow IPSP in the rabbit, but there maximum in about 0.8 msec and (Kobayashi and Libet, 1968; Nishi et is no general agreement on this point having a total duration of some 2.5 al., 1969) or in conjunction with an and norepinephrine may serve this msec. The generation of the inhibit­ increased resistance (frog) (Weight function in both the rabbit and other ory current is a consequence of a and Votava, 1970). The slow IPSP in mammalian species (Noon et al., brief increase in membrane conduc­ sympathetic ganglion cells from 1975; Lindl and Cramer, 1975; tance (Coombs et al., 1955; Smith et mammals evoked by synaptically re­ Eranko, 1976). In amphibia the slow al., 1967). The IPSP reverses from a leased catecholamines is also gener­ inhibitory transmitter is considered hyperpolarizing to a depolarizing po­ ated without any change in mem­ to be either acetylcholine (Weight tential at a membrane potential of brane resistance (Kobayashi and and Padjen, 1973b) or a about —80mV and was initially con­ Libet, 1968, 1970). catecholamine (Libet and sidered to be generated by increases Kobayashi, 1974). In both mammals Much of the evidence implicating in membrane permeability to potas­ and amphibians, the catecholamines cyclic nucleotides in synaptic trans­ sium and chloride ions. More re­ in ganglia which mediate the slow mission is derived from experiments cently, however, Lux has chal­ IPSP are present in small on sympathetic ganglia, and an ac­ lenged the assumption that chloride chromaffin-like cells possessing the count of the mechanisms underlying is passively distributed across the synaptic structures of interneurons the generation of slow ganglionic membrane, with an Eci near the (Matthews and Raisman, 1969; EPSP's and IPSP's is therefore par­ resting potential (—70 mV), and has Jacobowitz, 1970; Williams et al., ticularly relevant. The slow EPSP of proposed the existence of an out­ 1975; Eranko, 1976; Taxi and frog ganglion cells, in contrast to the wardly directed chloride pump in Mikulajova, 1976). fast EPSP which precedes it, is in­ motoneurons, with an Eci more neg­ creased by depolarization, de­ Nishi and Koketsu (1968) have ative than the resting membrane po­ creased by hyperpolarization, and proposed that the slow IPSP in frog tential (Lux et al., 1970; Lux, 1971). reverses into a hyperpolarization at sympathic ganglia is generated by an The evidence for this suggestion is a membrane potential close to the electrogenic sodium pump, as oua­ derived from the blockage of the potassium equilibrium potential bain, metabolic inhibitors and low chloride extruding mechanism by (Weight and Votava, 1970; but, see extracellular potassium depress the externally applied ammonium ions. Kobayashi and Libet, 1974). It was slow IPSP. Such a pump would ac­ Changes in chloride extrusion were postulated that the potential is gen­ tively extrude sodium ions from the

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ganglion cells, creating a net out­ as sodium, there is a tendency for II. IDENTIFICATION OF ward current, and thus hyper- the action potential to be reduced as SYNAPTIC TRANSMITTERS polarize them. This interpretation a result of compensatory movements The problem of transmitter iden­ has been disputed by Kobayashi and of these ions, which attempt to keep tification at a given synaptic junction Libet (1968) on the grounds that the membrane potential near their is a complex one and the criteria ouabain also depressed the fast own equilibria potentials. The net which have been developed have EPSP, and that depletion of ex­ result of both effects is to reduce the been discussed in detail elsewhere tracellular potassium did not selec­ amplitude of the presynaptic action (Werman, 1966; McLennan, 1970; tively depress the slow IPSP in rela­ potential and thus the amount of Phillis, 1970). Factors that must be tion to the EPSP. Weight and Pad- transmitter released (Schmidt, considered include: (1) that the sub­ jen (1973a) have proposed that the 1971). Presynaptic inhibition of this stance should be present in the ap­ slow IPSP in frog sympathetic gangl­ type is blocked by picrotoxin and propriate nerve endings together ion cells is generated by a decrease bicuculline, antagonists of the with the enzymes for its synthesis; in the resting sodium conductance, a monocarboxylic amino acid, (2) evidence for release of the sub­ mechanism which would account for y -aminobutyric acid (GABA). stance by impulses in the approp­ the reported increase in membrane GABA depolarizes nerve terminals riate nerve fibers; and (3) demonst­ resistance during the slow inhibitory and is therefore a likely candidate ration of a postsynaptic action that is potential. for the role of a presynaptic inhibit­ identical to that of the endogenously Other examples of slow synaptic ory transmitter. released transmitter, including selec­ transmission systems have been ob­ Somewhat different inhibitory tive antagonism by compounds act­ served in the cerebellar cortex and presynaptic actions may be as­ ing at the postsynaptic receptor. hippocampus. Fibers from the nuc­ sociated with other putative trans­ These criteria were developed for leus locus coeruleus hyperpolarize mitter agents. Extrinsic acetyl­ peripherally innervated junctions both Purkinje cells and hippocampal choline (ACh) significantly reduces and are rather more difficult to apply pyramidal cells by a mechanism ACh release from motor nerve ter­ at the complex junctions in the cen­ which is associated with an increase minals in skeletal muscle and at the tral nervous system. In practice, in membrane resistance (Hoffer et same time increases terminal excita­ neuropharmacologists have relied al., 1973; Segal and Bloom, 1974; bility (Ciani and Edwards, 1963; heavily on histochemical techniques Oliver and Segal, 1974). The ionic Hubbard et al., 1965). Similar effects for the localization of fiber tracts mechanism underlying these hyper- have been observed in sympathetic containing particular putative polarizations, or those evoked by ganglia (Nishi, 1970; Ginsborg, transmitters or the enzymes in­ norepinephrine, has still to be de­ 1971). In both muscle and ganglia the volved in their synthesis. Evidence termined. The actions of norepine­ presynaptic ACh receptors are on the release of transmitters in the phrine on neurons in the brain will nicotinic. In the central nervous sys­ CNS would be most meaningful if it be discussed in more detail in sec­ tem ACh may also have an inhibit­ were possible to detect and measure tion E. ory action on its own release (Szerb the release evoked at the appropriate (3) Presynaptic Inhibition and Somogyi, 1973) and on that of nerve terminals by a controlled, The potential importance of pre­ dopamine and norepinephrine brief stimulus applied to a homogen­ synaptic actions of transmitter (Reader et al., 1976) from the cere­ ous group of nerve cells of fibers. agents is becoming increasingly evi­ bral cortex. In this instance, the re­ This has not yet been achieved and dent to neuropharmacologists. As ceptors are muscarinic in type. with the techniques currently em­ originally postulated by Eccles Catecholamines act presynapti- ployed the transmitter is usually col­ (1963) for the cat spinal cord, pre­ cally in increasing ACh release at lected at some distance from the site synaptic inhibition was effected by a skeletal neuromuscular junctions of release and a relatively lengthy reduction in the amount of transmit­ (Krnjevic and Miledi, 1958; Jenkin- period of stimulation may be re­ ter released from a presynaptic son et al., 1968; Goldberg and quired to obtain detectable levels of nerve terminal during its invasion by Singer, 1969) but depress ACh re­ the transmitter. an action potential, when the termi­ lease from preganglionic nerve ter­ Due to the difficulties associated nal had itself been depolarized by a minals in sympathetic ganglia (Weir with intracellular recording from transmitter released from another and McLennan, 1963; Christ and central neurons it is often difficult to nerve terminal. Although depolari­ Nishi, 1971; Dun and Nishi, 1974). establish that the candidate transmit­ zation makes the affected terminals Nishi and his colleagues believe ter substance has an identical action more excitable, it also reduces the that the primary action of on membrane potential and conduc­ amplitude of action potentials invad­ catecholamines on sympathetic tance to that of the endogenously ing them by an amount at least equal ganglia is to decrease ACh output released transmitter. In practice to the amount of the depolarization. from presynaptic nerve terminals pharmacological tests, in which the Additionally, because the membrane and that their post-synaptic action, a candidate substance can be shown to permeability change which causes weak hyperpolarization, plays a be affected in an identical manner to the depolarization may involve secondary role in the depression of the synaptically released transmitter chloride and potassium ions as well ganglionic transmission. by the appropriate antagonists or

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potentiators, are often employed to brane or within the cell and thus 1976). Adenylate cyclase must there­ satisfy the criterion of identity of evoke the appropriate physiological fore contain sites for a divalent metal action. Although it has been as­ response. The cyclic nucleotides are and GTP as well as sites capable of sumed that only one transmitter is degraded by specific 3', 5'-phos­ binding with its substrate ATP (Rod- released at a given junction, it is phodiesterases to their respec­ bell et al., 1975). The mechanism by becoming increasingly possible to tive 5'-monophosphates. Ter­ which sodium fluoride is able to envisage nerve terminals which re­ mination of the physiological effects stimulate the enzyme is still uncer­ lease two or more substances having initiated by the cyclic nucleotides tain. significantly different actions (Burn- may occur by concurrent activation Cell free preparations of adenylate stock, 1976), in which case the of phosphoprotein phosphatases cyclase from brain tissue usually re­ criterion of identity of action could which hydrolyze phosphorylated spond minimally to neurotransmit­ not easily be applied. Similar dif­ proteins, thus restoring them to their ters (Rail and Sattin, 1970; Von ficulties may arise when putative original inactive state. Hungen and Roberts, 1974; Daly, transmitter substances activate more 1975; but see Chasin et al., 1974) and than one set of receptors on the (1) Adenylate Cyclase slices of brain tissue have therefore neuron. For instance, there is now This enzyme catalyzes the reac­ been utilized in most studies on evidence that ACh may have both tions in which ATP, in the presence receptor-mediated regulation of cyc­ excitant and depressant effects on ofMg++ , is converted to cAMP and lic AMP levels. corticospinal neurons, both of which pyrophosphate (Rail and Sutherland, are antagonized by the muscarinic 1962; Sutherland et al., 1962; (2) Guanylate Cyclase blocker, atropine (Bevan et al., Drummond and Ma, 1975). Brain Guanylate cyclase and cyclic 1975). contains high levels of adenylate GMP are present in brain and sym­ pathetic ganglia, although their Substances which are presently cyclases, associated with plasma levels are usually one order of mag­ considered to be potential neuro­ membranes (Perkins, 1973; Burkard, 1975). Levels are higher in gray than nitude lower than those of adenylate transmitters in the central nervous cyclase and cyclic AMP (Goldberg system include the mono- and di- in white matter and vary considera­ bly in different areas of the brain; the et al., 1970; Goldberg et al., 1973; carboxylic amino acids (glycine, Hardman and Sutherland, 1969; GABA, aspartate and glutamate), overall activity being high in the cerebral cortex and cerebellum Steiner et al., 1972; Kuo et al., 1972; acetylcholine, dopamine (DA), Ferrendelli et al., 1970; Ferrendelli norepinephrine (NE), epinephrine, (Klainer et al., 1962; Weiss and Costa, 1968; Ebadi et al., 1971; et al., 1972; Ferrendelli et al., 1973). serotonin (5-HT), histamine, sub­ Cyclic GMP levels are particularly Schmidt et al., 1971; Steiner et al., stance P, adenosine and adenine high in the cerebellum, where in 1972; Wellmann and Schwabe, nucleotides. The effects of these some rodents the amounts are nearly 1973). The enzyme is present in both agents on brain levels of cAMP and as high as those of cyclic AMP. A neuronal and glial-enriched fractions cGMP will be assessed in the next major portion of guanylate cyclase in section. from rat and rabbit brain (Palmer, brain homogenates appears to be 1973) and in cultured neuroblastoma membrane bound (Sulakhe et al., C. METABOLISM AND and glioma cells (Chlapowski et al., 1976), although activity is also pres­ FUNCTIONAL CHARACTER­ 1975). In brain homogenates, high ent in the cell cytoplasm. Manganese ISTICS OF CYCLIC levels of both cAMP and adenylate seems to be absolutely required for NUCLEOTIDES cyclase have been reported in frac­ brain guanylate cyclase basal activ­ tions enriched with nerve terminal I. COMPONENTS OF THE ity (Nakazawa and Sano, 1974), and CYCLIC NUCLEOTIDE membranes (De Robertis et al., in the presence of low levels of SYSTEM 1967; Johnson et al., 1973). Mn++ , Ca++ has a stimulant ac­ The nucleotides, cAMP and The activity of adenylate cyclase tion on the enzyme. Fluoride ions do cGMP, are formed from intracellular in brain homogenates is enhanced by not alter basal activity. ATP and GTP respectively, by the magnesium or manganese ions: cal­ action of cyclase enzymes (adeny­ cium ions may have either stimulant (3) Protein Kinases late cyclase or guanylate cyclase). or depressant actions. Sodium Cyclic AMP-dependent protein These cyclases are activated when fluoride has a stimulant action on the kinases in brain homogenates are the hormone or neurotransmitter at­ enzyme in cell free preparations but found in both soluble and particulate taches to its receptor on the cell not in whole cells (Bradham et al., fractions, including those enriched membrane, thus raising the intracel­ 1970; Perkins and Moore, 1971; Per­ with synaptosomes (Miyamoto et lular level of the cyclic nucleotide. kins, 1973; Von Hungen and al., 1969a and 1969b; Miyamoto et The diverse biological effects of the Roberts, 1974). 5'-guanylimido- al., 1971). These kinases catalyze nucleotides are manifested through diphosphate, an analogue of GTP the phosphorylation of serine re­ activation of a family of enzymes which stimulates adenylate cyclase sidues in a variety of protein sub­ known as protein kinases which can in many peripheral tissues, also strates including histones, glycogen phosphorylate other enzymes or stimulates adenylate cyclase in synthetase, synaptosomes and mic­ structural proteins in the cell mem­ monkey cerebral cortex (Ahn et al., rotubules (Goldberg and O'Toole,

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1969; Kuo and Greengard, 1969a, b, phosphodiesterase in rat cortical tiple forms of phosphodiesterase in 1970a, b; Miyamoto et al., 1969a, b; slices is localized at postsynaptic the brain with potentially variable Goldberg et al., 1970; Johnson et al., membranes on dendritic processes drug sensitivities will make it essen­ 1971; Maeno et al., 1971; Schmidt (Florendo et al., 1971), and in glial tial that a better understanding of and Sokoloff, 1973; Weller and Rod- cells (Shanta et al., 1966). the pharmacology of the indivi­ night, 1973). Phosphorylation of two The total phosphodiesterase activ­ dual isoenzymes be obtained before species of synaptic membrane pro­ ity in brain is a result of a variety of the effects of inhibitors on func­ tein (Protein I and Protein II) was different enzymes which vary in tional systems can be assessed in greatly increased by cyclic AMP their properties with regard to sub­ a meaningful manner. (Johnson et al., 1973; Ueda et al., strate specificity and affinity, elec- 1973). The cyclic AMP-activated (5) Phosphoprotein Phosphatases ++ trophoretic mobility and sensitivity If the phosphorylation of mem­ protein kinases require Mg or to cations (Kakiuchi et al., 1975; Mn++ and are inhibited by Ca++ . brane proteins is an essential event Amer and Kreighbaum, 1975). At in synaptic transmission, enzymes Cyclic GMP activates these kinases least two major forms of the en­ only at high concentrations. (phosphatases) should be present to zyme, one with a high affinity for restore the protein molecule to its Cyclic GMP-activated protein cyclic AMP and another with low original state at the termination of kinases appear to be present in brain affinity, occur in most regions of the the synaptic event. Phosphoprotein preparations and catalyze the phos­ brain. These two enzymes follow phosphatases have been found in the phorylation of histones (Kuo and different patterns of development in rat cerebral cortex (Maeno and Greengard, 1973; Kuo, 1974). These the rat (Weiss and Strada, 1972). Greengard, 1972; Ueda et al., 1973; kinases require Mg++ for activity Certain phosphodiesterases will ++ Maeno et al., 1975) and are concen­ and are inhibited by Ca . Cyclic catabolize both cyclic nucleotides, trated in the synaptic membrane- AMP-dependent protein kinases although they may show some containing subfractions. have been described in neuroblas­ specificity for one or the other sub­ toma (Greengard and Kuo, 1970) and strate, and the rate of catabolism is glioma cells (Perkins et al., 1971; often influenced by the level of the II. CRITERIA FOR ACCEPTANCE Opler and Makman, 1973). As in other cyclic nucleotide. OF CYCLIC NUCLEOTIDES other tissues, cyclic nucleotide- AS SECOND MESSENGERS Phosphodiesterase activity in the The criteria by which the synaptic activated kinases from brain are dis­ soluble fraction of brain homoge­ sociable into regulatory units which transmitter at a given junction can be nates is stimulated by low concent­ identified were discussed in Section bind cyclic AMP and the functional rations of Ca++ in the presence of a catalytic units (Inoue et al., 1973). B. Sutherland and his colleagues protein activator. In contrast to par­ (Sutherland et al., 1968; Robison et The relatively inactive complex of ticulate phosphodiesterases, which regulatory and catalytic units is dis­ al., 1971) established a series of four apparently hydrolyze cyclic AMP at criteria which must be satisfied be­ sociated and thereby activated in the a higher rate than cyclic GMP, the presence of cyclic AMP. fore a cyclic nucleotide can be ac­ soluble enzymes have a higher affin­ cepted as the mediator of hormonal ity for cyclic GMP than for cyclic (4) Phosphodiesterase ++ action. These criteria have been 3', 5'-Phosphodiesterases are the AMP. Ca release into the cyto­ adapted for use at synaptic junc­ enzymes which catabolize cyclic plasm may therefore be an important tions. nucleotides by splitting the 3' leg of factor in the regulation of cyclic First, cyclic nucleotide levels in the cyclic phosphate bond yielding GMP levels (Appleman and the postsynaptic cell should respond the 5'-purinoside monophosphate Terasaki, 1975; Kakiuchi et al., appropriately to stimulation of the (Appleman et al., 1973; Amer and 1975). afferent pathway or to the transmit­ Kreighbaum, 1975; Kakiuchi et al., Brain phosphodiesterases are in­ ter substance. (2) The change in cyc­ 1975). Levels of cyclic AMP phos­ hibited by a variety of compounds, lic nucleotide levels should precede phodiesterases fluctuate considera­ including methylxanthines such as the physiological response. (3) The bly between different brain regions theophylline, theobromine and caf­ effects of activation of the synaptic and between species (Weiss and feine, imidazole, papaverine, di­ pathway or of application of the Costa, 1968; Breckenridge and pyridamole, chlordiazepoxide, dia-; transmitter should be potentiated by Johnston, 1969; Dalton et al., 1974). zepam, tricyclic antidepressants, agents which inhibit phosphodies­ Phosphodiesterases have been found chlorpromazine and other pheno- terase activity. (4) It may be possible in both soluble and particulate frac­ thiazines, phentolamine, adenosine, in some instances to mimic the ef­ tions from brain homogenates (De RO 20-1724 (4-[3-butoxy-4-methoxyj fects of synaptic activation by the Robertis et al., 1967) and they are 2-imidazolidinone) and SQ 20,009 addition of exogenous cyclic AMP present in homogenates from cul­ (l-ethyl-4-isopropylidene hydrazino) or GMP. A further criterion pro­ tured neuroblastoma and glioma (lH-pyrazolo [3,4-0] pyridine-5- posed by Kuo and Greengard (1969a, cells (Weiss, 1975; Schwartz et al., carboxylate, ethylester) (Weiss, b; see Bloom, 1975) is that activa­ 1973; Uzunov et al., 1973). His- 1973; Amer and Kreighbaum, 1975; tion of a phosphotransferase reac­ tochemical studies have shown that Daly, 1975). The existence of mul­ tion will be the major mechanism by

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which cyclic nucleotides express ficulties are circumvented by the use D. FORMATION OF CYCLIC their effects. of synthetic nucleotides, which are NUCLEOTIDES IN THE CNS more lipid soluble and resistant to In practice, the first two criteria I. PREPARATIONS USED FOR are difficult to satisfy since the phosphodiesterase action (Simon et STUDIES ON CYCLIC methods employed for measuring al., 1973; Miller et al., 1973), there is NUCLEOTIDES cyclic nucleotides are usually ap­ the problem of uncontrolled access Cyclic nucleotide levels have been plied to tissues containing large of the nucleotides to multiple active studied in intact brain, brain slices, sites within the cell. Indeed, Borle numbers of cells and are not suffi­ brain homogenates and in cultured (1974) has shown that cyclic AMP ciently rapid to detect changes oc­ neuronal and glial cell lines. curring within the latencies of onset can stimulate Ca++ release from of synaptic potentials. Immuno- mitochondria in a variety of tissues (1) Investigations in vivo fluorescent assays of cyclic AMP and extracellularly applied cyclic Cyclic AMP levels in intact brain and cyclic GMP which enable semi­ AMP or an analog could thus affect tissue rise rapidly after decapitation quantitative measurements of cyclic cell excitability through a Ca++ - (Breckenridge, 1964; Kakiuchi and nucleotide formation in individual dependent mechanism quite inde­ Rail, 1968a), and rapid fixation by nerve cells to be made should offer pendently of any transmitter-related microwave irradiation (Schmidt et some prospect of satisfying the de­ actions. al., 1971) or freeze-blowing (Lust et al., 1973) is required. Cyclic GMP mands of these criteria (Bloom et al., In summary, it can be stated that levels in intact brain do not undergo 1972; Kebabian et al., 1975). The complete satisfaction of the criteria such marked changes on decapita­ third criterion — potentiation of the is extremely difficult and that even tion (Goldberg et al., 1970; Steiner et effects of the synaptic pathway by the satisfaction of criteria (3) and (4) phosphodiesterase inhibitors — has is fraught with hazards. The pres­ al., 1972). The factors involved in been extensively employed in ence of cyclic AMP in almost every post-decapitation increases in cyclic studies on the cyclic nucleotides, but cell has led to the now widely ac­ AMP levels in brain are not as yet it too is not without its hazards. cepted view that this compound firmly established, but it is probable Many of the agents which are cur­ must have a universal role as a sec­ that anoxia leads to an accumulation rently used as phosphodiesterase in­ ond messenger. It is difficult to of adenosine, which then stimulates hibitors have other potent actions avoid the conclusion that in their the formation of cyclic AMP (Berne which may be responsible for the enthusiasm to accept this view, et al., 1974). effects observed. The methylxan- many investigators have not dis­ Several investigators have re­ thines, caffeine and theophylline, ++ played a sufficiently cautious ap­ ported on the alterations in cyclic cause Ca release from the sar­ proach to the interpretation of their nucleotide levels in brain in the pre­ coplasmic reticulum of muscle and data. There can be little doubt that sence of agents, including possibly from the plasma membrane many agents which affect nerve cell catecholamines and D-lysergic acid (Isaacson and Sandow, 1967; John­ excitability also alter their cyclic diethylamide, which affect the cen­ son and Inesi, 1969; Blinks et al., nucleotide levels. The question that tral nervous system excitability 1972; Berridge, 1975). Effects of must be asked is "Do these altera­ (Chou et al., 1971; Burkard, 1972; these agents may result from in­ tions in cyclic nucleotide levels Uzunov and Weiss, 1972; Opmeer et creases in intracellular calcium cause (or mediate) the elec­ al., 1976). levels rather than elevation of cyclic trophysiological effects of the ap­ However, the difficulties as­ AMP levels. Theophylline and caf­ plied agent or do there merely occur sociated with post-decapitation in­ feine also block an extracellularly in parallel with, or as a result of, the located adenosine receptor (Huang creases in cyclic AMP referred to electrophysiological effects?" The above have hindered the interpreta­ and Daly, 1974). Papaverine, another role of calcium ions as second mes­ extensively utilized phosphodies­ tion and comparison of data ob­ sengers has been largely overlooked tained from studies on intact brain terase inhibitor, prevents the uptake as investigators concentrated on the of adenosine, and may interfere with and from experiments on brain slice cyclic nucleotides. In subsequent preparations, the experimental pre­ cell excitability by potentiating the sections attention will be focussed actions of adenosine, either released paration favored by most inves­ on the potential role of this ion as a tigators. from adjacent tissues or formed from mediator of the effects of neuro­ exogenously applied cyclic AMP. transmitters, and on the possibility (2) Brain Slice Preparations Interpretation of the effects of ex­ of a cooperative interaction between The ability of certain putative ogenously applied cyclic nucleotides Ca+ " and the cyclic nucleotides neurotransmitters to enhance cyclic may also be difficult. Cyclic nuc­ (Rasmussen, 1970; Rasmussenet al., AMP, and to a lesser extent cyclic leotides do not readily cross cell 1972; Borle, 1973; Berridge, 1975). GMP, levels in brain slices has been membranes and, when applied to the documented by a number of inves­ exterior of the cell, are exposed to tigators. Cyclic nucleotide accumu­ the action of phosphodiesterases lation has been measured either by which may catabolize them to direct assay or by determination of 5'-nucleotides. Even when these dif­ the extent of radioactive cyclic nuc­ leotide formation from labelled pre-

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cursors such as adenine or glioma cell lines respond to species or for different areas of the adenosine. Adenine or adenosine are norepinephrine with remarkable in­ brain: Each tissue appears to have incorporated into intracellular nuc­ creases in cyclic AMP formation. its own pattern of responsiveness. leotides by the action of adenine In one study with rat glioma cells, The experiments on cyclic AMP phosphoribosyltransferase or the maximal responses to formation have yielded valuable in­ adenosine kinase respectively. norepinephrine were not observed formation about the nature of the Brain slices from different regions until many days after the cells had neurotransmitter receptors in brain of the brain and a variety of species become confluent (Schwartz et al., slices and have provided specific have been investigated, and this 1973), while in another study with agonists and antagonists which have preparation has undoubtedly pro­ human glioma cells the maximal re­ been used to elucidate the role of the vided a valuable indication of the sponses to norepinephrine were at­ corresponding cyclic AMP-systems factors involved in the regulation of tained before the cells had formed a in the intact brain. A limitation of the brain nucleotide levels. confluent mono-layer, and declined studies on brain slices is that unless after further culture (Clark and Per­ they are combined with prior de­ (3) Cells from Neuronal or Glial Cul­ kins, 1971). struction of selected pathways, they tures do not yield a clear understanding of The brain slice contains multiple (4) Cell-free Preparations of Adeny­ whether the transmitter-activated cell types and investigations utilizing late Cyclase cyclases are located in pre- or post­ this preparation have yielded results Klainer et al. (1962), in one of the synaptic structures or even in glial which are difficult to ascribe to any early reports on brain adenylate cyc­ cells. These experiments therefore one particular cell type. For this lase, stated that activity in brain serve only to establish the existence reason, the use of cell cultures has cell-free preparations could be of receptor-coupled cyclases; they become an attractive alternative. stimulated by various define neither the site nor the nature Cultures have been prepared from catecholamines. Stimulation was of any physiological responses reaggregated systems derived from most often observed in cerebellar which result in or from the accumu­ fetal mouse or rat brain, although preparations, but was variable and lation of cyclic AMP. these probably still contain rela­ inconsistent. Until recently, at­ Neurochemists have been tempted tively normal proportions of neurons tempts to reproduce these findings to conclude that cyclic AMP plays a and glia (Seeds, 1971). Results ob­ were largely unsuccessful, and it is pivotal role in the generation of the tained from pure neuronal and glial apparent that the adenylate cyclase inhibitory responses to cell cultures must be interpreted system in brain tissue is especially catecholamines, serotonin and his­ with some caution since the tumor prone to loss of sensitivity to neuro­ tamine, throughout the central ner­ cell lines from which these are de­ transmitters as a result of tissue dis­ vous system. This conclusion is rived may show greatly altered en­ ruption. However, methods of brain based, however, on satisfaction of zyme levels or responsiveness to homogenization that yield vesicular but the first of the criteria outlined in neurotransmitters compared to that preparations which retain section C, namely that the transmit­ of the parent tumor cell or normal receptor-modulated adenylate cyc­ ter can influence the formation of cells. For example, clones of glioma lases have been reported (Chasin et cyclic AMP. Further critical inves­ cells often lose their responsiveness al., 1974; Von Hungen et al., 1974). tigations will be required to establish to various agents with time (Perkins, Cell-free preparations differ from the conclusion's general validity. 1973). The age of the culture may be those containing intact nerve cells in (1) Norepinephrine that fluoride ions activate adenylate significant for other reasons. Cell Norepinephrine causes an ac­ cultures from fetal or embryonic cyclase in the former, although they cumulation of cyclic AMP in all the brain may be relatively refractory have no effect on intact cells (Per­ areas of the brain on which it has during the early period of their reag- kins, 1973). Fluoride may act at the been tested, including cerebral cor­ gregation to agents which elevate catalytic site of the enzyme, causing tex, cerebellum, limbic system, cyclic AMP levels in mature brain stabilization as well as activation of hypothalamus and caudate nucleus. preparations. For instance, em­ adenylate cyclase activity (Swislocki The observed increases in basal bryonic mouse brain cells do not and Tierney, 1973). adenylate cyclase activity range respond to norepinephrine with an from just detectable to up to 20 fold increase in cyclic AMP at 15 hours II. NEUROTRANSMITTERS in the guinea pig cerebellum. Phar­ when aggregation was essentially AFFECTING CYCLIC macological investigations have re­ complete, though the response is NUCLEOTIDE FORMATION vealed that the adrenergic receptors manifest at 9 days (Schmidt et al., The mass of information on in brain do not fit the classical 1970). These results are reminiscent transmitter-stimulation of cyclic definitions of peripheral a - and of the finding that norepinephrine nucleotide formation in brain slices (3 -receptors, since in most regions does not stimulate cyclic AMP for­ has recently been reviewed by Daly of the brain both alpha and beta mation in rat brain slices from ani­ (1975, 1976) and presented in tabular blockers are partial antagonists. The mals under 4 days of age. form by Bloom (1975). It is difficult extent of activation of cyclic AMP At certain times during culture to make generalizations across generating systems by ex - or

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)3 -receptor interactions appears to on adenylate cyclase in presynaptic phodiesterase inhibitor (Schultz and differ markedly in the same brain terminals (Von Hungen and Roberts, Daly, 1973b; Dismukes and Daly, area of different species and, in addi­ 1974). It is of some interest therefore 1974; Daly, 1975). Serotonin elicits a tion, varies greatly in different brain that the existence of adenylate significant 2-fold increase in cyclic regions of the same species. Cere­ cyclase-coupled a -receptors on AMP levels in the squirrel monkey bral cortical responses to norepineprhine-containing nerve polysensory cortex, an area contain­ norepinephrine range from nearly terminals has been proposed (Dis- ing multiple serotonergic nerve end­ pure a -adrenergic in the guinea pig mukes and Mulder, 1976). Activa­ ings (Skolnick et al., 1973). Cell free (Huang et al., 1971), to a mixture of tion of these receptors markedly re­ preparations from the superior and a - and (3 -adrenergic in the rat duces norepinephrine release from inferior colliculus of very young rats (Huang et al., 1973; Perkins and rat brain slices. contain adenylate cyclase systems Moore, 1973) to nearly pure Norepinephrine stimulates both which are highly responsive to /3 -adrenergic in mouse and man cyclic AMP and cyclic GMP forma­ serotonin. The response diminishes (Schultz and Daly, 1973a; Shimizu et tion in mouse cerebellar slices at with age and is relatively insignifi­ al., 1971). The /3 -agonist, iso­ concentrations as low as 1 u M (Fer- cant in collicular preparations of proterenol, has only one half the rendelli et al., 1975). The accumula­ adult rats (Von Hungen et al., 1975). maximal effect seen with norepine­ tion of cyclic GMP, but not of cyclic phrine in rat cortical slices, and its (4) Histamine AMP, is blocked by omission of effects are completely blocked by ++ Histamine-sensitive cyclic AMP Ca from the incubation medium. /3 -antagonists but unaffected by generating systems have been re­ The receptor sites mediating the a -antagonists. In cerebellar slices ported in slices from a variety of cyclic nucleotide accumulation from rat, guinea pig or rabbit, the species and brain regions, including responses to norepinephrine are appear to have both a - and the cerebral cortex, cerebellum, completely blocked by /? -adrenergic sensitivities. hippocampus and thalamus (Daly, (2) Dopamine 1975; Hegstrand et al., 1976). The (3 -antagonists, while ot -antagonists Dopamine-sensitive adenylate responses appear to be mediated are ineffective (Skolnick et al., 1976; cyclases have been reported in through both Hi and H: receptors Chasin et al., 1971; Kakiuchi and homogenates or slices from the although the relative importance of Rail, 1968b). Neuroleptics such as caudate nucleus, nucleus accumbens the two receptor types may vary. fluphenazine reduced the response and olfactory tubercle (Clement- For instance, in the guinea pig cere­ of rat cerebellar cortex to Cormier et al., 1974; Clement- bral cortex the responses are norepinephrine by 50 - 60 percent Cormier et al., 1975; Horn et al., mediated through an interaction with (Hoffer et al., 1976). Norepinephrine 1974; Iversen, 1975), and cerebral both Hi and H2 receptors (Baudry may also activate dopamine- cortex (Dismukes and Daly, 1974; et al., 1975; Dismukes et al., 1976), sensitive adenylate cyclases such as Nahorski and Rogers, 1976; Ahn et while in the rat and chicken cortex H2 those present in slices of caudate al., 1976). receptors predominate (Dismukes et nucleus (Forn et al., 1974). High (1 mM) concentrations of al., 1975; Nahorski et al., 1974). Studies on cultured cells of dopamine elevate cyclic AMP and Histamine elicits cyclic AMP ac­ neuronal or glial origin have shown depress cyclic GMP levels in mouse cumulations in certain glioma cell that norepinephrine and dopamine cerebellar slices (Ferrendelli et al., lines (Clark and Perkins, 1971). Cyc­ can markedly stimulate the accumu­ 1975). Dopamine-stimulated adeny­ lic AMP levels in neuroblastoma lation of cyclic AMP in some late cyclases are present in various cells are unaffected by histamine, neuroblastoma clones (Prasad et al., neuroblastoma cell lines (Sahu and even in the presence of phosphodies­ 1974; Sahu and Prasad, 1975; Prasad, 1975). terase inhibitors (Gilman and Niren- Schubert et al., 1976). Norepine­ berg, 1971; Schultz and Hamprecht, phrine also elicits cyclic AMP and Systemically administered apo- cyclic GMP formation in various morphine, a direct dopaminergic 1973; Prasad et al., 1974). glioma cell lines (Perkins, 1973; agonist, elevates cyclic GMP levels in rat and mouse cerebella (Burkard et (5) Adenosine Daly, 1975; Schwartz, 1976; Adenosine elicits an accumulation Schubert et al., 1976). al., 1976; Gumulka et al., 1976). Mus­ carinic antagonists block this effect in of cyclic AMP in slices from all brain Immunocytochemical studies on rats but not in mice, implying the exist­ regions and virtually all species the rat cerebellar cortex have shown ence of a cholinergic link in the rat. evaluated (Rail and Sattin, 1970; Sat- that topically applied norepine­ tin and Rail, 1970; Daly, 1975, 1976). phrine or stimulation of nore- (3) Serotonin Adenosine's action is shared by the pinephrine-containing afferents Serotonin elicits small accumula­ adenine nucleotides such as from the locus coeruleus cause a tions of cyclic AMP in rabbit cere­ 5'-AMP, ADP, ATP and cyclic AMP striking increase in cyclic AMP bral cortical and cerebellar slices (Sattin and Rail, 1970; Rail and Sat­ levels in the postsynaptic neurons, (Kakiuchi and Rail, 1968a, 1968b). tin, 1970), which are thought to un­ the Purkinje cells (Siggins et al., In guinea pig and rat cerebral cortex dergo extracellular hydrolysis to 1973). There is less information re­ its effects are minimal except in the adenosine in order to exert their garding the effects of norepinephrine presence of adenosine and a phos­ effects. The stimulatory effects of

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ATP and cyclic AMP on adenylate the excitant amino acids and de­ brain homogenates (Duffy et al., cyclase are nearly completely polarizing agents are partially an­ 1975). blocked by adenosine deaminase. As tagonized by theophylline, indicat­ (7) Amino Acids ATP and cyclic AMP are not sub­ ing that adenosine release within the Cyclic AMP and cyclic GMP for­ strates for this enzyme it is evident slice is involved (Shimizu et al., mation in cerebral and cerebellar that prior hydrolysis to adenosine is 1974; Daly, 1975; Mah and Daly, brain slices is stimulated by the ex­ necessary before they can activate 1976). Sattin et al. (1975) recently citant dicarboxylic amino acids, the cyclic AMP generating system proposed the idea that adenosine L-glutamic acid and L-aspartic acid (Mah and Daly, 1976). The observa­ and a catecholamine or histamine (Shimizu et al., 1974; Chasin et al., tion that adenosine can elicit an may act as either dependent or inde­ 1974; Ferrendelli et al., 1974; accumulation of cyclic AMP from pendent co-activators of brain Kinscherf et al., 1976). The inhibit­ pre-labelled stores of intracellular adenylate cyclases. ory monocarboxylic amino acids, ATP provides evidence that Adenosine and its analogs act as GABA and glycine, elicited a small adenosine stimulates adenylate cyc­ potent activators of adenylate cyc­ decrease and a small increase re­ lase by activation of an adenosine lase in neuroblastoma and glioma spectively in cyclic AMP levels in receptor (Daly, 1975), rather than cell lines (Clark and Gross, 1974; mouse cerebellar slices, and both acting as a precursor for substrate Blume and Foster, 1975; Daly, 1975; agents enhanced cyclic GMP levels ATP. This concept of an adenylate Clark and Seney, 1976; Penit et al., in mouse cerebellum, but not in cyclase-coupled adenosine receptor 1976; Green and Stanberry, 1977). cerebral or cerebellar slices from is strengthened by the finding that rabbit, guinea pig, cat or rat (Fer­ certain analogs of adenosine, such as (6) Acetylcholine and substance P rendelli et al., 1974; Kinscherf et al., 2-chloro-adenosine, stimulate ac­ There are relatively few reports .1976). The stimulatory effects of the cumulation of cyclic AMP but do not on the effects of ACh or other excitant amino acids on cyclic AMP serve as precursors for the cyclic cholinomimetic agents on cyclic levels are reduced under Ca++ -free nucleotide thus formed (Sturgill et AMP levels in brain slices or other in conditions or in the presence of the al., 1975). Fairly conclusive evi­ vitro preparations. An ACh- adenosine antagonists, theophylline dence for the extracellular location sensitive adenylate cyclase has been and 3-isobutyl-l-methylxanthine, sug­ of the receptor was obtained in demonstrated in cultured neuroblas­ gesting that a depolarization-induced studies with compounds which in­ toma cells (Prasad et al., 1974; Sahu release of adenosine accounts for hibit adenosine uptake into tissue, and Prasad, 1975). In rabbit cerebral part of their action (Ferrendelli et including dipyridamole, hexoben- and cerebellar cortical slices, ACh al., 1974; Shimizu et al., 1974), dine, papaverine and £-nitro- and muscarinic agonists reportedly Glutamate and the biogenic amines phenylthioguanosine. These agents elicit an increase in cyclic GMP lev­ have synergistic effects on the for­ enhance the response to low els (Lee et al., 1972; Kuo et al., mation of cyclic AMP in guinea pig concentrations of adenosine, while 1972; Palmer and Duszynski, 1975). cortical slices. concomittantly inhibiting the incor­ The effects were antagonized by poration of adenosine into the tissue atrophine. However, other inves­ The stimulatory effect of the by 70 - 90 percent (Huang and Daly, tigators, using brain slices from cor­ amino acids on cyclic GMP accumu­ 1974). One striking property of the tex and cerebellum of several lation requires the presence of adenosine receptors is that they are species, have been unable to repli­ theophylline (Ferrendelli et al., competitively blocked by the cate these observations (Kinscherf 1974). Mao et al. (1974a, b) have methylxanthine phosphodiesterase et al., 1976). Parenterally adminis­ reported that a relationship may inhibitors, including theophylline, tered oxotremorine, a muscarinic exist between GABA and cyclic caffeine and 3-isobutyl-l-methylxan- agonist, causes increases in cyclic GMP in rat cerebellum. Drug treat­ thine (Huang et al., 1972; Mah and GMP in the mouse cerebral cortex ments in vivo which lowered GABA Daly, 1976). and cerebellum, with a concomitant levels were associated with an in­ decrease in cyclic AMP levels. At­ crease in cyclic GMP, and in- Adenosine displays a remarkable tracerebroventricular injections of synergism with various other agents ropine prevents these effects of ox­ otremorine on cyclic GMP levels GABA decreased cerebellar cyclic with respect to enhancement of cyc­ GMP levels. lic AMP formation in brain slices. (Ferrendelli et al., 1970). Substances which have synergistic Substance P, a polypeptide pres­ (8) Prostaglandins actions with adenosine include the ent in brain, spinal and some prim­ Although the role of prostaglan­ biogenic amines, norepinephrine, ary afferent fibers, excites neurons dins in intercellular communication serotonin and histamine (Sattin and at several levels of the neural axis, in the central nervous system is pre­ Rail, 1970; Schultz and Daly, 1973b; and may be the transmitter released sently uncertain, a variety of find­ Mah and Daly, 1976) glutamate and at the central terminals of some dor­ ings suggest that prostaglandins of aspartate (Shimizu et al., 1974) and sal root afferent fibers (Nicoll, 1976; the E series do play a significant part depolarizing agents such as potas­ Phillis, 1977). Preliminary reports in central nervous function (Siggins sium, veratridine, batrachotoxin and indicate that substance P activates et al., 1971b). Prostaglandins have ouabain (Daly, 1975). The effects of adenylate cyclase in rat and human been reported to have no effect on

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cyclic AMP levels in brain cortical to this theoretical train of events stimulated dopamine or norepine­ slices and homogenates from a vari­ cyclic nucleotide levels would be phrine release from internuncial ety of mammals, as well as on restored to normal by the action of cells within the ganglion. This norepinephrine-stimulation of cyclic phosphodiesterases and the mem­ catecholamine caused both the slow AMP formation (Forn and Krishna, brane potential would be restored to IPSP and the related increase in cyc­ 1971; Palmer et al., 1973; Zanella resting levels by the dephosphoryla- lic AMP. Immunocytochemical ex­ and Rail, 1973). However in two tion of the membrane proteins and periments on isolated bovine ganglia investigations prostaglandins Ei and return of normal resting membrane have provided further support for E2 elicited accumulations of cyclic permeability. It is assumed that the existence of separate AMP in rat cortical slices (Berti et these events occur sufficiently nucleotide-linked muscarinic and al., 1972; Kuehl et al., 1972). In a rapidly to account for the time dopaminergic receptors on the recent study, prostaglandin Ei was course of the postsynaptic potential. post-ganglionic neurons. Dopamine found to elicit significant accumula­ and norepinephrine increased cyclic tions of cyclic AMP in slices from rat I. SYMPATHETIC GANGLIA AMP but not cyclic GMP levels in cortex, midbrain and hippocampus Much of the evidence in support slices of these ganglia and the in­ (Dismukes and Daly, 1975). Little or of roles for cyclic AMP and cyclic crease was localized to the post­ no stimulating effect was seen in GMP in synaptic transmission has ganglionic neurons. Acetylcholine slices from cerebellum or pons- been obtained from studies on the increased cyclic GMP but had only a medulla. Studies with combinations mammalian superior cervical gangl­ slight effect on cyclic AMP levels in of prostaglandin Ei and norepine­ ion. This preparation is particularly the postganglionic neurons (Kebabi­ phrine suggested that prostaglandin suitable for experiments of this na­ an et al., 1975). The absence of the Ei antagonized the /3 -adrenergic ture since it can be isolated and anticipated effect of acetylcholine component of the norepinephrine re­ studied under well-controlled condi­ and bethanecol on cyclic AMP levels sponse and at the same time elicited tions using precise electrophysiolog­ in these experiment was attri­ an accumulation of cyclic AMP ical techniques for stimulation and buted to damage to the catechola- (Dismukes and Daly, 1975). Prostag­ recording. Also the tissue can be mine-containing interneurons during landins Ei and E2 activate adenylate fixed rapidly by freezing for the preparation of the tissues. cyclase in neuroblastoma cell lines biochemical analysis. Electrical stimulation of the preganglionic (Gilman and Nirenberg, 1971; Ham- The responses of bovine superior fibers produces an increase in the precht and Schultz, 1973; Sahu and cervical ganglia to dopamine are in­ cyclic AMP and cyclic GMP levels Prasad, 1974; Penit et al., 1976) and hibited by a -antagonists but not by in the ganglion (McAfee et al., 1971; in certain glioma cell lines (Minna /3 -antagonists. /3 -Antagonists do, Weight et al., 1974; Greengard and and Gilman, 1973; Perkins, 1973). however, reduce the effects of Kebabian, 1974). The muscarinic an­ norepinephrine (Kebabian and tagonist, atropine, blocks this in­ Greengard, 1971). In contrast, cyclic E. NUCLEOTIDES AND TRANS­ crease in the levels of both nuc­ AMP levels in cultured rat superior MISSION AT SELECTED leotides, suggesting that it occurs cervical ganglion are elevated by SYNAPSES postsynaptically as the result of ac­ norepinephrine and isoproterenol, tivation of muscarinic receptors by while even higher concentrations of Support for a function of the cyc­ ACh released from the presynaptic lic nucleotides in synaptic transmis­ dopamine or phenylephrine have nerve terminals. The increase in cyc­ only marginal effects. The effects of sion has come from studies on the lic AMP and cyclic GMP content can distribution of the enzymes and pro­ norepinephrine are antagonized by be mimicked by the application of both a - and (1 -adrenergic blockers teins involved in the formation, de­ agents capable of activating mus­ gradation and function of cyclic (Cramer et al., 1973). The increases carinic receptors such as carbachol, in rat superior cervical ganglion cyc­ AMP and cyclic GMP. The enzyma­ bethanecol, and in the case of cyclic tic machinery associated with the lic AMP induced by isoproterenol GMP, acetylcholine (Kalix et al., are lost when the ganglionic cells are metabolism of the cyclic nucleotides 1974; Kebabian et al., 1975). seems, at least in part, to be as­ destroyed by 6-hydroxydopamine sociated with the synaptic mem­ (Otten et al., 1974), indicating that Reference was made in Section B the isoproterenol-sensitive cyclases brane subfraction of brain homoge­ of this paper to the evidence that nates, as are the cyclic nucleotide- are located in adrenergic cell bodies. the slow EPSP and slow IPSP in Basal levels of cyclic AMP are only dependent protein kinases. The mammalian sympathetic ganglion theory predicts that certain mem­ slightly reduced in ganglia from cells are mediated by muscarinic and 6-hydroxydopamine treated rats. brane proteins are phosphorylated dopaminergic (or noradrenergic) re­ by cyclic nucleotide-stimulated pro­ ceptors respectively. The evidence Greengard and his colleagues have tein kinases and that phosphoryla­ suggested that acetylcholine re­ obtained electrophysiological evi­ tion of these proteins leads to the leased from the presynaptic nerve dence to support their hypothesis membrane permeability changes terminals evoked the slow EPSP by that the effects of acetylcholine and causing the postsynaptic excitatory an action at muscarinic receptors on catecholamines are mediated by cyc­ or inhibitory potentials. According the postganglionic cell bodies and lic GMP and cyclic AMP respec-

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tively, by studying the effects of actions of 1-2 mM dibutyryl cyclic concentration-dependent increase in these agents on synaptic potentials AMP on the resting potential or on membrane conductance (Shinnick- and changes in membrane potential action potentials (Kuba and Nishi, Gallagher et al., 1976). Since the of the rabbit superior cervical gangl­ 1976). Similar results were obtained slow IPSP and slow EPSP of gangl­ ion using the sucrose gap technique with sucrose gap techniques by ion cells are generated in the ab­ (McAfee and Greengard, 1972; Akasu and Koketsu (1977). Dun and sence of any decrease in membrane Greengard, 1976). Preganglionic Karczmar (1977) have compared the conductance (Section B), even these stimulation of the ganglion resulted effects of theophylline and cyclic findings with intracellularly injected in the generation of an initial brief AMP on the superior cervical gangl­ nucleotides cannot easily be related EPSP, followed by a slow IPSP that ion of the rabbit with the sucrose gap to synaptic events. reached a maximum in 600 msec and method. Neither cyclic AMP nor di­ In studies on parasympathetic then a slow EPSP lasting about 30 butyryl cyclic AMP, in concentra­ ganglia of the urinary bladder of the sec. Exogenous dopamine (50 \x m) tions of up to 2.5 mM, had any cat, for which an adrenergic inhibit­ hyperpolarized the postganglionic noticeable effect on membrane po­ ory mechanism has been described, neurons; an effect which was poten­ tential or on the compound action de Groat and Theobald (1976) ob­ tiated by theophylline. Theophylline potential elicited by preganglionic served minimal depression of gang­ also increased the amplitude of the stimulation. In curarized prepara­ lionic transmission with cyclic AMP slow IPSP. Prostaglandin Ei (PGEi), tions cyclic AMP did not affect the or dibutyryl cyclic AMP injections. which is considered to inhibit adeny­ positive (P) or late negative (LN) Non-cyclic nucleotides, in smaller late cyclase, virtually abolished the potentials, although the P wave was doses, produced considerable de­ slow IPSP and reduced the slow potentiated by theophylline. These pression of ganglionic transmission. EPSP, but had little effect on the investigators concluded that their These substances also depressed fast EPSP. At a concentration of 0.1 findings were inconsistent with the bladder contractions elicited by elec­ p. M PGEi also abolished or reduced hypothesis of a cyclic AMP-related trical stimulation of the pregang­ the dopamine-evoked hyperpolariza- mechanism in the generation of lionic nerves, but did not alter the tions. Monobutyryl cyclic AMP(1 to ganglionic inhibitory potentials and responses to injected acetylcholine, 2.5 mM) hyperpolarized the ganglia; suggested that the effects of raising the possibility that they had a similar results were obtained with theophylline might involve presynaptic effect and depressed cyclic AMP and dibutyryl cyclic mechanisms other than phos­ bladder contractions by preventing AMP, but not with 5'AMP, phodiesterase inhibiton. Using more the release of acetylcholine. Non- adenosine or butyric acid, which refined techniques, Dun (personal cyclic adenine nucleotides depress were inactive. Dibutyryl cyclic GMP communication) has applied cyclic the release of ACh from skeletal (25-250 u M) caused a transient AMP and dopamine iontophoreti- motor nerve terminals (Ginsborg and hyperpolarization followed by a de­ cally onto ganglion cells and re­ Hirst, 1972), and further experimen­ polarization in each of the prepara­ corded the responses intracellularly. tation will be required to ascertain tions tested. Cyclic GMP itself, No cyclic AMP-induced hyper- whether they have a similar effect in however, was inactive. polarizations were detected in cells sympathetic ganglia. The findings of these studies sup­ that were hyperpolarized by In summary, it is difficult to avoid port the concept of a role of cyclic dopamine. Similar results have been the conclusion that the roles of cyc­ AMP and cyclic GMP in ganglionic obtained by Libet, Kobayashi and lic AMP and cyclic GMP as second transmission. The evidence indi­ Tanaka (personal communication) messengers at synapses on sym­ cates that cyclic GMP is involved in who comment, "Superfusing the pathetic ganglion cells are less con­ the generation of the slow EPSP and rabbit superior cervical ganglion vincingly established than they ap­ cyclic AMP is involved in the gener­ with dibutyryl cyclic AMP or peared to be a few years ago. Re­ ation of the hyperpoiarizing re­ monobutyryl cyclic AMP at 1-2 mM ports from several laboratories have sponse to dopamine. Cyclic GMP for 6-10 minutes, has rarely elicited failed to confirm the initial observa­ and cyclic AMP thus appear to func­ any hyperpoiarizing response. In the tions of a hyperpoiarizing action of tion in opposite directions, one en­ so-called positive instances, the re­ cyclic AMP and a depolarizing ac­ hancing and the other depressing sponses were inconsistent and were tion of cyclic GMP on ganglion cells. neuronal excitability in the ganglion. probably mostly if not entirely an Until these discrepancies have been Unfortunately other investigators artefact of the sucrose gap techni­ resolved, the evidence in favor of a have not been able to reproduce que." second messenger role of cyclic nuc­ these effects of the cyclic nuc­ Extracellular applications of di­ leotides in ganglia must be consi­ leotides on sympathetic ganglion butyryl cyclic AMP and dibutyryl dered suspect. Pending further in­ cells, using either sucrose gap or cyclic GMP had no effect on the vestigations, it would be unwise to intracellular recording techniques. intracellularly recorded membrane regard sympathetic ganglia as Intracellular recordings from the properties of rat superior cervical proven examples of nucleotide paravertebral sympathetic ganglia of ganglion cells, but when cyclic AMP mediated synapses and for the pres­ the bullfrog (Rana catesbiana) have or cyclic GMP were injected in­ ent it must be assumed that the post­ failed to reveal any hyperpoiarizing tracellularly, both caused a synaptic accumulations of cyclic

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nucleotides either result from or characterizing the receptors as (h) Cerebral Cortex occur in parallel with the synaptic 0 -adrenergic. The /S -adrenore- Norepinephrine has an inhibitory events. ceptor has now been visualized on action on cerebral cortical neurons Purkinje cell bodies and apical in several species (Krnjevic and Phil- II. CENTRAL "AMINERGIC" dendrites in rat cerebellum by a TRANSMISSION lis, 1963; Lake et al., 1973) including new fluorescent analogue of pro­ identified pyramidal tract neurons Norepinephrine-containing axons pranolol (Melamed et al., 1976). (Phillis, 1970; Stone, 1973). Both a- arising in the pontine nuclei, locus Stimulation of the nucleus locus and /? -adrenergic blockers an­ coeruleus and sub-coeruleus, extend coeruleus has an inhibitory action on tagonize this depression. to the cerebral, cerebellar and limbic Purkinje cell and hippocampal Norepinephrine-induced hyper- cortices (Ungerstedt, 1971; Olson neuron firing which is also blocked polarization of feline cortical and Fuxe, 1971; Pickel et al., 1974). by Sotalol (Hoffer et al., 1973; Segal neurons generally occurs in the ab­ Synaptic terminals presumed to con­ and Bloom, 1974b). Intracellular re­ sence of any pronounced alteration tain norepinephrine have been iden­ cording has revealed that both in membrane resistance (Fig. I). tified in the rat cerebellar cortex, norepinephrine and locus coeruleus Electrophysiological evidence for a where the principal target neurons stimulation hyperpolarize the target monosynaptic projection to the cor­ are thought to be the Purkinje cells cells in the cerebellar cortex and tex from the nucleus locus coeruleus (Bloom et al., 1971). There is also hippocampus, often with an as­ has been obtained in experiments on anatomical and histochemical evi­ sociated increase in membrane resis­ the antidromic activation of locus dence that cells in the cerebral tance (Hoffer et al., 1973; Oliver and coeruleus neurons by cortical stimu­ cortex and pyramidal neurons Segal, 1974). The latency of the in­ lation (Nakamura and Iwama, 1975; of the hippocampus receive nore­ hibition in Purkinje cells averaged Faiers and Mogenson, 1976). The pinephrine-containing afferents 148 msec, implying conduction vel­ antidromic latencies for activation of from the locus coeruleus (Ungers­ ocities in the afferent fibers as low as locus coeruleus neurons by frontal tedt, 1971; Descarries and LaPierre, 0.075 - 0.1 m/sec. cortical stimulation ranged from 1973; Pickel et al., 1974). Similar 20-70 msec, with conduction vel­ lines of evidence indicate that After chronic pretreatment of the ocities estimated to be 0.4 - 1.3 dopamine-containing axons from the animals with 6-hydroxydopamine, M/sec. pars compacta of the substantia which destroys catecholaminergic nigra synapse extensively on cells of neurons, stimulation of the nucleus Studies on identified corticospinal the caudate nucleus (Hokfelt and locus coeruleus failed to evoke sig­ and unidentified deep spontaneously Ungerstedt, 1973). In a separate pro­ nificant inhibition of Purkinje cell or firing neurons have revealed an in­ jection, dopamine-containing fibers hippocampal pyramidal cell dis­ hibitory action of nucleus locus arise from neurons in the substantia charges. Similar results were ob­ coeruleus stimulation (Fig. 2) (Phillis nigra and ventro-medial tegmentum tained in animals which were treated and Kostopoulos, 1977). The in­ and project to the rhinencephalic with reserpine, which depletes hibitory effects of norepinephrine cortex, the temporal cortex and the catecholamine stores in brain, to­ and locus coeruleus stimulation on anterior cingulate gyrus (Lindvall et gether with alpha-methyltyrosine, a these neurons were antagonized by al., 1974; Berger et al., 1974). norepinephrine synthesis blocker the /3 -adrenergic blocker, Sotalol (Hoffer et al., 1973; Segal and Serotonin is concentrated within (Fig. 3), and pretreatment of the Bloom, 1974b). neurons of the brain stem raphe nuc­ cerebral cortex with lei which are the source of serotonin-containing fibers to the NA 200 R forebrain, including the hippocam­ D sec pus and cerebral cortex (Anden et iiiJiiiiliinlniiliiiJiUiyMiiliMyiMtlMtilniiliiiiliiiiliiiiLiiliiiiliiiiliililiiMln.iliiip mliliillillMMiiilm||i al., 1966; Conrad et al., 1974; Kuhar et al., 1972; Lorens and Goldberg, Or 1974). (1) Norepinephrine-Mediated mV Synapses (a) Cerebellar Purkinje Cells and Hippocampal Pyramidal Neurons Iontophoretically applied norepinephrine depressed the spon­ Figure I—A. Intracellular potential of a cerebral cortical neuron recorded on a chart recorder. Norepinephrine (NA, 200nA) hyperpolarized the neuron. 0.5 nA pulses of taneous firing of Purkinje cells and inward and outward current lasting 250 msec were used to test the neuron's input hippocampal pyramidal neurons. impedance. This did not change during the norepinephrine-induced hyperpolariza- The inhibition in both instances is tion. B. Recording made after the microelectrode had been withdrawn from the blocked by the /3 -adrenergic an­ neuron showing the almost negligible effects of electrode polarization and bridge tagonist Sotalol (MJ 1999) (Hoffer et imbalance. Time calibrations: I and 5 sec. (J. P. Edstrom and J. W. Phillis. al., 1971; Segal and Bloom, 1974a), unpublished observation).

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make extensive synaptic contacts with various groups of motoneurons. The actions of iontophoreticaliy ap­ plied norepinephrine are likely therefore to be mediated via synap­ VK***. j* tic receptors. (2) Dopamine-Mediated Synapses A nigro-striatal dopamine contain­ /Aryy-yytf ing projection has been demon­ UvfV/'V^V^ strated by biochemical and cytochemical techniques (Hor- nykiewicz, 1966; Hokfelt and Un- gerstedt, 1973; York, 1975). Ion­ tophoreticaliy applied dopamine in­ hibits the firing of caudate neurons in cats and rats (Siggins et al., 1974) Connor, 1975). These effects of dopamine are antagonized by the phenothiazines, chlorpormazine, fluphenazine and alphaflupenthixol, Figure 2—A. B and C. Records from three cerebral cortical neurons. B and C were and are mimicked by apomorphine identified by antidromic activation as corticospinal neurons. The records in (1) and (Siggins et al., 1974). The dopamine (2) are time histograms recorded during repetitive stimulation of the locus coeruleus receptor in the caudate was not an­ (3 pulse train) and in the absence of LC stimulation respectively. The records in (3) tagonized by the /3 -adrenergic an­ are recordings of neuronal firing rate and show that norepinephrine (NA, 80 nA) tagonist, Sotalol (Siggins et al., depressed the discharge rate of all three neurons. Ordinate calibrations: (1) and (2), 1974). number of neuronal discharges; (3) neuronal firing rate as spikes per second. The horizontal bars in (3) represent the periods of NA application. (From J. W. Phillis The effects elicited from caudate and G. K. Kostopoulos. 1977). units by nigral stimulation have proven to be rather variable. In a study of 260 feline caudate neurons 6-hydroxydopamine, which destroys Some of the terminals of these path­ in which firing had been induced by the norepinephrine-containing nerve ways make close contacts with the the application of an excitatory terminals, abolishes the inhibitory cell bodies and processes of spinal amino acid, 46 per cent were de­ effects of locus coeruleus stimula­ motoneurons. Fluorescent his- pressed, 16 per cent were excited, tion. The latencies of onset of locus tochemical studies have identified a and the remainder were unrespon­ coeruleus-evoked inhibition were bulbospinal serotonergic pathway sive to short trains of stimuli ap­ 32 ± 1.5 msec (mean ± S.E.M.) for which arises from cell bodies in the plied to the substantia nigra (Connor, corticospinal neurons and 54 ± 2.3 caudal raphe nuclei and gives rise to 1970). Neurons depressed by nigral msec for unidentified neurons. terminals at all levels of the spinal stimulation were consistently de­ When trains of 3 to 4 pulses were cord (Dahlstrom and Fuxe, 1965; Pin pressed by dopamine and alpha- applied to the locus coeruleus, the et al., 1968; Coote and MacLeod, methyldopamine antagonized the inhibition had a mean duration of 1974). depressant responses induced by 148 msec. Norepinephrine and serotonin both nigral stimulation and ion- These findings, in conjunction hyperpolarize motoneurons (Phillis tophoretic dopamine. In a study of with those from anatomical, et al., 1968a; Engberg et al., 1974) rat caudate neurons, a good correla­ neurochemical and release (Tanaka with a concomitant increase in tion between the inhibitory effects of et al., 1976) studies, argue convin­ membrane resistance. There is a substantia nigra stimulation and the cingly for an inhibitory nore- failure of invasion of ortho- and an- inhibitory effects of dopamine was pinephrine-mediated pathway from tidromically evoked spikes into the observed. Papaverine and bul- the locus coeruleus to the cerebral initial segment, soma-dendritic bocapnine applied iontophoreticaliy cortex. membrane and both EPSP's and or given intravenously blocked the (c) Spinal Motoneurons IPSP's are reduced. Both a - and effects of nigral stimulation and The locus coeruleus projects to /3 -blockers antagonize the effects of dopamine (Gonzalez-Vegas, 1974). the spinal cord (Kuypers and Man- norepinephrine, as do the neurolep­ These data suggest that the depres­ sky, 1975) and both this and other tics, chlorpromazine and sant responses elicited from many brain stem nuclei may contribute to haloperidol. caudate neurons by nigral stimula­ the descending catecholaminergic Although the effects of these de­ tion are mediated by a slowly con­ pathways in the spinal cord (Dahl- scending aminergic pathways have ducting monosynaptic dopaminergic strom and Fuxe, 1965; Pickel et al., still to be established, the anatomical pathway. Evidence for an excitatory 1974; Coote and MacLeod, 1974). data suggest that they probably dopaminergic projection from the

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The hippocampus receives a di­ rect input from the raphe nuclei (Conrad et al., 1974) and its seroto­ nin content is dependent on the in­ tegrity of the raphe nuclei (Kuhar et al., 1972; Lorens and Goldberg, 1974). Hippocampal pyramidal cells in the rat are inhibited by serotonin and by electrical stimulation of the dorsal and median raphe nuclei 100r (Segal, 1975). The inhibitory re­ sponse to raphe stimulation is absent in rats that have been pretreated with p -chlorophenylalanine, a serotonin synthesis inhibitor, and (2) MJ 1999 60 the effects of P -chlorophenylala­ nine are alleviated by administration of 5-hydroxytryptophan, the precur­ sor of serotonin. The inhibitory ef­ fects of both serotonin and raphe stimulation are blocked by the sero­ tonin antagonists, methysergide and cyproheptadine, and potentiated by chlorimipramine, a serotonin re­ uptake blocker. Serotonin therefore deserves serious consideration as an inhibitory transmitter acting on rat hippocampal pyramidal cells. OL Comparable responses have been observed in the rat sensory-motor cortex. Serotonin depresses these neurons and its effects are selec­ tively antagonized by the drug metergoline (Sastry and Phillis, 1977a). Metergoline also antagonizes the inhibitory response generated by raphe stimulation. The inhibitory ef­ fects of serotonin and raphe stimula­ J tion are enhanced by the selective serotonin uptake blocker (3-) P-tri- 50msec fluoromethylphenoxy)-N-methyl-3- phenylpropylamine (Lilly 110140) Figure 3—MJ-1999 antagonism of inhibition of the firing of a corticospinal neuron (Sastry and Phillis, unpublished ob­ induced by repetitive (3 stimuli) stimulation of the ipsilateral locus coeruleus. The upper and lower traces are poststimulus histograms recorded before and 6 min. after servations; Frederickson et al., an application of MJ-1999 (60 nA). The middle trace was recorded 3 min. after the 1975). onset of the MJ-1999 application, and shows that this B -adrenergic blocker (4) Histamine-Mediated Synapses antagonized the inhibitory effects of LC stimulation. MJ-1999 also antagonized the inhibitory effects of norepinephrine on this neuron. (From J. W. Phillis and G. K. Histamine and L-histidine decar­ Kostopoulos, 1977). boxylase, the enzyme which con­ verts L-histidine to histamine, are present in rat cerebral cortex substantia nigra to the caudate, and Phillis, 1963; Bunney and Agha- (Schwartz, 1975). Various biochem­ which is antagonized by chlor- janian, 1976), but the physiological ical studies suggest that a significant promazine, has recently been pre­ significance of the recently disco­ portion of the amine and its synth­ sented by Kitai et al. (1976), and this vered dopaminergic projection to esizing enzyme are associated with pathway may account for the ex­ this region of the brain remains to be the synaptosomal fraction of brain citatory effects of nigral stimulation evaluated (Lindvall et al., 1974; tissue (Kataoka and De Robertis, on caudate neurons. Berger et al., 1974; Fuxe et al., 1967; Garbarg et al., 1976). Transec­ Dopamine depresses neuronal fir­ 1974). tion of the rat medial forebrain bun­ ing in the cerebral cortex (Krnjevic (3) Serotonin-Mediated Synapses dle results in a reduction in his-

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taminergic pathway projects to the cerebral cortex. Discovery of the G nucleus of origin of these his­ 100r taminergic fibers will be keenly awaited.

III. DEPRESSANT ACTIONS OF CYCLIC AND NON-CYCLIC ADENINE NUCLEOTIDES (1) Cerebellar Purkinje Cells According to the criteria for ac­ ceptance of cyclic nucleotides as H second messengers outlined in Sec­ 100 tion C. II., it may be possible to mimic the effects of synaptic activa­ tion by the addition of exogenous cyclic nucleotide. It is the satisfac­ tion of this criterion, as it is in the case of the sympathetic ganglion, which has given rise to much of the controversy regarding the role of cyclic nucleotides in synaptic func­ tion in the central nervous system. Simply stated the problem is as fol­ lows: In the hands of Bloom and his colleagues cyclic AMP has been a consistent and potent depressant of neurons that were depressed by norepinephrine. In the experiences Figure 4— Metiamide (METI, 50 nA) effects on inhibitions of a deep cerebral cortical of many other investigators, cyclic neuronal firing induced by stimulation of the ipsilateral medial forebrain bundle AMP has proven to be a weak and (MFB, A. B and C) and the ipsilateral cortical surface (CS, D, E and F), as well as by inconsistent depressant of NE- iontophoretically applied histamine (HA. 50 nA, G and H). A and D illustrate the sensitive neurons. What is the ex­ control post-stimulus histograms for the cortical neuronal firing after MFB and CS planation for this crucial discrep­ stimulations, respectively. B was recorded 4 min. to 6 min. 8 sec. following the onset ancy! Various technical considera­ of METI application, whereas C was recorded 7 min. 30 sec. to 12 min. 50 sec. after tions must be taken into account in initiation of METI ejecyion. C and F depict a recovery from METI antagonism (C: 8 any attempt to analyze this problem. min. to 10 min. 9 sec. and F: 11 min. to 16 min. 20 sec. following the termination of METI application). G-H portrays a continuous recording of the neuronal firing rate. In order to assess the relative In G. HA (50 nA) decreased the discharge rate of this neuron and METI (50 nA) potencies of iontophoretically ap­ antagonized this depression. H shows a recovery from METI antagonism. Ordinate plied substances it is necessary to calibration: A-F: no. of neuronal discharges; G-H: neuronal firing rate as spikes per determine the transport number of second. The horizontal bars in G and H indicate the duration of drug applications. the compounds, which relates the (B. S. R. Sastry and J. W. Phillis. 1976b). amount of substance released from the micropipette and the magnitude famine levels and L-histidine decar­ cortical neurons, and a component and duration of the iontophoretic boxylase activity in the ipsilateral of this inhibition is reproducibly an­ delivery current. The transport cerebral cortex, suggesting that a tagonized by the Fh histamine number for cyclic AMP, determined histaminergic pathway may ascend blocker, metiamide (Fig. 4) (Sastry with ejection currents and delivery through this tract (Garbarg et al., and Phillis, 1976b). A substantial periods approximating those used in 1976). portion of the medial forebrain bun­ in vivo experiments (Shoemaker et Histamine inhibits the firing of dle evoked inhibition is resistant to al., 1975), was found to be margi­ cerebral cortical neurons (Phillis et metiamide, but this is not surprising nally lower than that for synaptic al., 1968b) and studies with Hi and as there is evidence that transmitter substances (0.05 versus Fh agonists and antagonists have serotoninergic and catecholaminer- 0.1 or more, Kelly, 1975). In addi­ provided evidence for the existence gic pathways ascend to the cortex tion a large amount of variation in of both Hi and H: receptors on cere­ through this pathway. Metiamide release was found within individual bral cortical neurons (Phillis et al., does not affect the inhibitory actions cyclic AMP-containing electrodes 1975; Sastry and Phillis, 1976a). of these amines. These results, to­ and among various pipette barrels, Stimulation of the medial forebrain gether with the neurochemical find­ but this is often even more pro­ bundle inhibits the firing of cerebral ings, suggest that an inhibitory his­ nounced for norephinephrine-

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containing barrels (Kelly, 1975). In minute period and when the neuron 3', 5'-cyclic nucleotides of adeno­ 1974, in response to the repeated then did not respond by a change in sine also suggests that catabolism to failures of other investigators to re­ rate of at least 10 per cent to at least 2'- or 5'-AMP and/or adenosine may plicate their data with cyclic AMP two consecutive pulses of 200 nA be an important factor in the ac­ on cerebellar Purkinje cells (God- each applied for at least 1 minute." tions of these compounds. 2', 5'-cyc- fraind and Pumain, 1971; Lake and In my own laboratory, 10 per cent lic AMP is hydrolyzed by a highly Jordan, 1974) Bloom and his col­ changes in firing rate are not re­ active and specific phosphodies­ leagues disclosed an apparently crit­ ported as significant, especially in terase located in myelin in the brain ical step in their technical proce­ the case of a cerebellar Purkinje cell, (Drummond et al., 1971). As 2', 3'- dures, that of electrode "warm-up". since these neurons are exquisitely cyclic phosphodiesterase is approxi­ This procedure requires the passage sensitive to current effects. How­ mately 2-10 times more active com­ of large (150-200 nA) currents for ever, the criteria described above pared to 3', 5'-cyclic phosphodies­ fairly lengthy periods of time (5 min demand that, to be considered as a terase (Drummond and Yamamoto, or more) through the cyclic AMP- negative, the neuron should NOT 1971; Drummond and Ma, 1975), the containing barrel prior to its actual change its firing rate by more than 10 hydrolysis of 2', 3'-cyclic AMP is use of Purkinje cells. "Warm-up" is per cent in two consecutive trials. It likely to proceed at a considerably a remarkable phenomenon since it is rather astonishing therefore to find higher rate than that of 3', 5'-cyclic apparently induces a long-lasting in­ that in the above investigation only AMP. The low potency of extra- crease in the transport number for 63 per cent of the Purkinje cells cellularly applied 3', 5'-cyclic AMP cyclic AMP in any particular elec­ tested were depressed by cyclic may thus be a reflection of a low AMP. In an investigation conducted trode barrel, an effect that has not rate of hydrolysis to 5'-AMP. This in this laboratory, where cyclic AMP been reported by other inves­ concept also explains the low poten­ was applied by currents of up to 80 tigators, even though it is known cy of extracellularly applied dibuty- nA for periods of up to 2 min, 37 per that the amount of iontophoretic re­ ryl 3', 5'-cyclic AMP, as this com­ cent of the Purkinje cells were de­ pound is resistant to catabolism by lease is affected by the strength and pressed (Kostopoulos et al., 1975). phosphodiesterases (Drummond and duration of the previous retention A comparable percentage of de­ current (Bradshaw et al., 1973; pressed Purkinje neurons was re­ Powell, 1970). The possibility that Clarke et al., 1973). There is a possi­ ported by Lake and Jordan (1974), exogenous cyclic AMP may act as a bility that with these large who used currents of up to 200 nA. precursor for adenosine has been "warm-up" currents, the current noted by other investigators looking density and power dissipation in the Regardless of the parameters of at its pharmacological actions on region of the electrode tip could application used, the crucial obser­ smooth muscle, and metabolic ef­ cause accelerated oxidation or hyd­ vation apparent in all these results is fects on cultured human lympho- rolysis of drugs in the electrode, that cyclic AMP is a rather weak blasts (Kim et al., 1968; Snyder and including a breakdown of cyclic depressant of the firing of some Pur­ Seegmiller, 1976). AMP to the much more potent kinje cells. Of further concern is the The role of cyclic AMP in 5'-AMP (see below). Another possi­ possibility that exogenously applied norepinephrine inhibition has been bility that must be considered is that cyclic AMP may be converted to questioned on the grounds that the electro-osmotic movements of fluid 5'-AMP and adenosine and that its percentage of Purkinje cells de­ into the pipette will occur during the pharmacological actions are actually pressed by this agent does not match passage of the "warm-up" currents mediated by these compounds. Sig­ the high percentage depressed by and these may carry soluble phos­ gins et al. (1971a) observed only NE. This difference has been attri­ phodiesterases into the electrode, excitant actions when 5'-ATP and buted to technical factors relating to again resulting in the formation of 5'-AMP were tested on Purkinje cyclic AMP release from the mic- 5'-AMP from cyclic AMP. cells, although a weak depressant ropipette and to the necessity that effect of adenosine was subse­ cyclic AMP (a sparingly permeable- It is interesting to have a detailed quently reported (Bloom et al., agent) reach intracellular protein look at the criteria used by Siggins 1975). In another study, however, kinases, through the regulation of and Henriksen (1975) in a recent pronounced depressant actions of which it can cause the phosphoryla­ report on the effects of cyclic AMP several adenine nucleotides, includ­ tion of membrane proteins and thus on rat Purkinje cells. In addition to ing adenosine 2', 3'-monophosphate, alter the biophysical properties of the normal conditions of current were reported (Kostopoulos et al., the cell membrane. To overcome 1975). neutralization and constant spike this latter difficulty, various amplitude it is stated that "Since the If non-cyclic nucleotides have analogues which are more potent interpretation of negative data with very much more potent actions that cyclic AMP itself in activating iontophoresis is difficult, a classifi­ than cyclic AMP, extracellular brain protein kinases were tested on cation of 'no effect' was given a cell catabolism of the latter to 5'-AMP Purkinje cells. The only when the drug barrel had been and/or adenosine must be consi­ 8-parachlorophenylthio- and 'warmed up' by several pulses of at dered. The marked difference in 8-benzylthio- analogues of cyclic least 100 nA applied over a 2 to 5 the potencies of the 2', 3'- and AMP inhibited the firing of a higher

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TABLE 1 Depressant Potency of Various Purines and Pyrimidines on Corticospinal Cells

Pronounced Weak Very Weak or Nil Adenosine Adenosine Adenine Adenosine 2'-monophosphate 3'. 5'-cyclic monophosphate Cytidine Adenosine 3'-monophosphate Guanosine 5'-monophosphate Cytidine 3' (2')-monophosphate Adenosine Guanosine 5'-diphosphate Cytidine 5'-monophosphate 2'. 3'-cyclic monophosphate Guanosine 5'-triphosphate Cytidine 5'-triphosphate Adenosine 5'-monophosphate Guanosine Guanosine Adenosine 5'-diphosphate 3'. 5'-cyclic monophosphate Hypoxanthine Adenosine 5'-triphosphate Inosine Adenosine 5'-tetraphosphate Inosine 5'-monophosphate Inosine 5'-triphosphate Purine riboside Thymidine Thymidine 5'-monophosphate Thymidine 5'-triphosphate Uridine Uridine 3' (2')-tnonophosphate Uridine 5'-monophosphate Uridine 5'-triphosphate Xanthine Xanthosine Xanthosine 5'-monophosphate Xanthosine 5'-triphosphate

percentage of neurons than did cyc­ lic AMP (Siggins and Henriksen, 5-ATP 20 5-ADP20 5-AMP20 NA30 1975), strengthening the link be­ tween adrenergic inhibition of Pur- kinje cells and its intracellular medi­ ation by cyclic AMP. However, 35r when tested on NE-depressed neurons in the cerebral cortex, these compounds had virtually no depres­ sant activity (Phillis and Edstrom, 1976), a difference which is difficult to explain. (2) Cerebral Cortical Neurons An extensive range of purine and pyrimidine nucleotides has been tested on identified corticospinal and deep spontaneously firing neurons in the rat sensory-motor cortex (Table 1). Adenine nucleotides and adenosine were observed to have a pronounced depressant action, guanine nucleotides a weak depres­ sant action, and little effect was observed with the cytidine, inosine, uridine and xanthine nucleotides (Phillis et al., 1974). Further studies have confirmed the potent depres­ 0L sant actions of 2'-, 3'-, 5'- L J adenosine monophosphates and 2', 3'-cyclic adenosine monophos­ 1min phate, and the relatively weak de­ Figure 5—Potent depression of spontaneous firing of a corticospinal neuron by adenine pressant action of 3', 5'-cyclic AMP nucleotides compared with weaker inhibitory action of norepinephrine (NA). Ratemeter record of neuronal firing. Application currents are shown in nA. (Fig. 5; Table 2) (Phillis et al., 1976).

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Substitution of halogen groups in plain their high potency and long 5'-AMP. As uptake into adjacent position 2 of the adenine ring of duration of action. cellular elements represents adenosine gave analogs which were The importance of adenosine an alternative mechanism for either significantly more potent than deaminase in terminating the action the removal of adenosine from the adenosine (2'-chloro- and 2'-fluoro- of adenosine was investigated with extracellular spaces, studies were in­ adenosine, Fig. 6) or which, in the two potent inhibitors of this enzyme, itiated with uptake blockers, includ­ case of 2-bromoadenosine, had a deoxycoformycin and erythro-9- ing papaverine, hexobendine and very prolonged duration of action. (2-hydroxy-3-nonyl) adenine. Both 2-hydroxy-5-nitrobenzylthioguano- 2-substituted adenosine derivatives substances caused a pronounced sine. Potentiation of the actions of are poor substrates for adenosine potentiation of the duration of de­ adenosine and 5'-AMP by these deaminase and this factor may ex­ pression evoked by adenosine and compounds was observed. Hex­ obendine had a potent depressant action of its own on the spontaneous firing of cortical neurons, a property TABLE 2 which complicated its use as an up­ Structure-Activity Relationships for Adenosine Analogs take blocker. The structure-activity relation­ Depressant Stimulation of ships displayed in Table 2 are re­ Activity Cyclic AMP markably consistent with those re­ on Cerebral Formation in sulting from biochemical investiga­ Cortical Cerebral Cortical Substance Neurons a Slices b tions on the stimulation of cyclic AMP formation in brain cerebral cortical slices by adenosine analogs. Adenine — 0 Adenosine + + ++ As stated previously, these biochem­ Inosine 0 ical results have been interpreted as being indicative of the existence of Purine Derivatives of Adenosine an extracellular receptor, stimula­ 2-Chloroadenosine -+f + ++ tion of which by adenosine evokes 2-Fluoroadenosine + ++ cyclic AMP formation within the 2-Bromoadenosine cell. The similarities between the 2-Aminoadenosine + ++ agonist potencies of various 2-Hydroxyadenosine + +-f- adenosine analogs observed in our 8-Bromoadenosine 0 0 0 experiments and the biochemical Ribose Derivatives of Adenosine studies suggest that in both in­ 2'-Deoxyadenosine 0 stances, a similar adenosine receptor 3'-Deox3'-Deoxyadenosiny adenosinee 0 is involved. Further support for a 5'-Deox5'-Deoxyadenosiny adenosinee +-+ one receptor hypothesis has been Adenine xylofuranosidxylofuranosidee — 0 forthcoming from studies with Adenosine N'-oxide — adenine derivatives on the depres­ Adenosine 5'-sulphat5'-sulphatee 0 0 sion of the amplitude of evoked Adenosine 5'-mononicotinat5'-mononicotinatee 0 postsynaptic potentials and simul­ Phosphorylated Derivatives taneous formation of cyclic AMP in Adenosine 5'-monophosphat5'-monophosphatee + + ++ guinea-pig olfactory cortex slices Adenosine 5'-diphosphat5'-diphosphatee + 4+- (Kuroda et al., 1976a). Both actions Adenosine 5'-triphosphat5'-triphosphatee + + + of adenosine can be prevented by Adenosine 5'-tetraphosphat5'-tetraphosphatee + + + elevated calcium levels in the solu­ Adenosine 2'-monophosphat2'-monophosphatee + ++ tion perfusing the slices (Kuroda et Adenosine 3'-monophosphat3'-monophosphatee 4+- ++ al., 1976b) and it has been proposed Adenosine 3', 5'-cyclic monophosphatmonophosphate +++ + ++ that adenosine depression of evoked Adenosine 2'. 3'-cyclic monophosphatmonophosphate postsynaptic potentials results from Adenosine 5'-imidodiphosphate inhibition of the action potential in­ Homoadenosine 6'-phosphonic acid 0 Oi, /3/3 -Methylen-Methylene 5'-AT5'-ATP —— 00 itiated calcium influx into the pre­ 0,7 -Methylene 5'-ATP 0 synaptic nerve terminal. a,/3-Methylene 5'-ADP — 8-Parachlorophenylthio 3', 5'-cAMP — Methylxanthines such as caffeine 8-Benzylthio 3', 5'-cAMP 0 0 and theophylline antagonize the 2-Deoxyadenosine 5'-monophosphate adenosine-evoked formation of cyc­ a Data from Phillis and Kostopoulos (1975); Phillis and Edstrom (1976). lic AMP in brain slices. When tested b Data from Sattin and Rail (1970); Huang et al. (1972); Huang and Daly (1974) and Mah and on cerebral cortical neurons, ion- Daly (1976). tophoretically applied caffeine

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AMP AMP 2-FIA 2-FIA adenosine-like action on cortical 20 30 20 20 neurons (Phiilis and Edstrom, 1976), but later biochemical studies on 501— neuroblastoma cells and ventricular myocardium have shown that 2'- and 3'-deoxyadenosine can stimulate endogenous cyclic AMP formation (Green and Stanberry, 1977; Huang and Drummond, 1976). The question of whether prior de­ gradation of the adenine nucleotides to adenosine is necessary before they can activate the receptor has been resolved with the use of 0l_ four methylene isosteres of 5'-AMP, B -ADP and -ATP. The phosphorus methylene bond is stable and resis­ 3or tant to metabolic transformations involving either hydrolysis or phos­ phate transfer (Yount, 1975). The a , /3 -isosteres of ADP and ATP were very weak depressants of cor­ tical neurons, in contrast to the j3 ,y - analog of ATP which posses­ sed quite pronounced depressant ac­ tivity (as did 5'-adenyl-imidodi- phosphate) (AMP-PMP) (Fig. 8). This finding suggested that even though the terminal bond in the ATP analogs was stable, cleavage could still occur at the a , Q position. 1 min The methylene analog of 5'-AMP, Figure 6—Depression of the spontaneous firing of a cerebral cortical neuron by homoadenosine 6'-phosphonic acid, adenosine S'-monophospha^e (AMP), 2-fluoroadenosine (2-FIA) and had no depressant activity on 2-chloroadenosine (2-ClA). Rate meter records of neuronal firing rate with the cerebral cortical neurons (Fig. 8) in­ number of action potentials per second on the ordinate. Bars indicate periods of drug dicating an absolute requirement for application. Application currents are shown in nA. (From J. W. Phiilis and J. P. dephosphorylation to adenosine for Edstrom, 1976). expression of depressant activity. Similar findings have been obtained blocked the depression of spontane­ Daly (1976) have shown that the in studies on brain slices, where ous firing induced by adenosine and stimulant action of exogenous cyclic Q! , /3 - and /J , 7 -methylene ATP 5'-AMP. Theophylline is so spar­ AMP on endogenous cyclic AMP had only a weak effect on cyclic ingly soluble as to be difficult to formation by brain slices is absent in AMP formation (Mah and Daly, apply by iontophoresis. This agent the presence of adenosine 1976). Reference was made earlier (and caffeine) was therefore tested deaminase. Cyclic AMP is not a sub­ to the lack of depressant effects of by systemic (intravenous) adminis­ strate for this enzyme and its action 8-benzylthio-cyclic AMP on cerebral tration. In doses of 10-100 mg/kgm on adenylate cyclase must be de­ cortical neurons. This compound theophylline (and caffeine) blocked pendent on a prior hydrolysis to also failed to enhance cyclic AMP the effects of adenosine and adenosine or 5'-AMP. formation in guinea-pig cerebral cor­ 5'-AMP. The antagonism could be Other antagonists of the tical slices. overcome if larger amounts of adenosine-elicited accumulation of adenosine were applied, implying cyclic AMP in brain slices are 2'-, 3'- An insight into the mechanism by that theophylline may act as a com­ and 5'-deoxyadenosine. Initial re­ which adenosine depresses the spon­ petitive antagonist. Theophylline ports (Mah and Daly, 1976) sug­ taneous firing of rat cerebral cortical also antagonized the depressant ac­ gested that the 2'- and 3'- com­ neurons has been gained by intracel­ tions of cyclic AMP (Fig. 7) confirm­ pounds lacked agonist action and lular recording with extracellular ing the suggestion that this cyclic could thus be considered as pure drug application (Phiilis and Ed­ nucleotide can evoke its effects antagonists. It was somewhat per­ strom, 1976; Edstrom and Phiilis, through activation of the extracel­ plexing therefore to discover that 1976). For technical reasons these lular adenosine receptor. Mah and these compounds had a weak studies were conducted with

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5'-AMP and the most striking find­ 5-AMP 20 AMP ings were that 5'-AMP caused a 3.5-CAMP60 cAMP hyperpolarization of the postsynap­ tic neurons in the absence of any significant alteration in membrane 50 r resistance, but with a decrease in spontaneous and evoked postsynap­ tic potentials. The mechanism by which 5'-AMP produces these ef­ fects is still by no means clear, but seems to involve some form of inter­ ference with the synaptic 0L mechanism. It may involve an inhib­ ition of presynaptic excitation- THEOPH. B AMP cAMP cAMP transmitter release coupling, as has 50mg/kg — been shown at peripheral cholinergic and adrenergic synapses (Ginsborg and Hirst, 1972; Miyamoto and 50r Breckenridge, 1974; Hedquist and Fredholm, 1976). Another possibil­ ity is that it may block the post­ synaptic receptors and (or) inhibit the coupling between the receptor and the conductance mechanisms controlled by the receptor. Hyper- polarizing actions of cyclic AMP on cerebellar Purkinje and hippocampal cells have been described (Hoffer et al., 1973; Oliver and Segal, 1974), AMP but these were associated with an AMP cAMP cAMP increase in membrane resistance. 50 T (3) Other Regions of the Brain

Adenosine and 5'-AMP have been tested on neurons that were iden­ tified by electrophysiological and/or histological means in the olfactory bulb, thalamus, hippocampus, caud­ ate nucleus and superior colliculus. 0L Neurons in each area were depress­ ed in a similar fashion to cerebral cortical neurons (Fig. 9). Compari­ 1min sons based on the proportion of units depressed by 5'-AMP and adenosine Figure 7— Theophylline antagonism of 5'-AMP(20 nA) and 3', 5'-cyclic AMP(60nA) and of the average application cur­ depression of the spontaneous firing of a corticospinal neuron. Theophylline (50 rents needed to induce a 50% reduc­ mg/kg) was administered intravenously after the control nucleotide responses had tion in firing rate showed that the been recorded. Trace B starts 7 min. after the theophylline injection when both 5'-AMPand 3', 5'-cyclic AMP depressions were abolished. Trace C was recorded 75 relative sensitivities of neurons in min. after theophylline when the 5'-AMP responses were beginning to recover. 3'. the different regions tested was: 5'-cyclic AMP was still blocked. hippocampus >. cerebral cortex > caudate and thalamus > superior colliculus > olfactory bulb. Ade­ this compound than cerebellar Pur­ motoneurons in the feline spinal nosine and 5'-AMP-depressed kinje cells (Siggins et al., 1974). cord in the expectation that when synaptically evoked firing in the Cyclic AMP also depressed neurons administered at this locus it should superior colliculus and olfactory in the cat brain stem (Anderson et more readily be able to initiate its bulb was as susceptible to adenosine al., 1973), nucleus accumbens (Bun- postulated "second messenger" ac­ and 5'-AMP-induced depression as ney and Aghajanian, 1973) and en- tion (Krnjevic and Van Meter, 1976). was the spontaneous firing. topeduncular nucleus (Obata and The most striking effects were a Cyclic AMP has a depressant ac­ Yoshida, 1973). speeding-up of the action potential, tion on caudate neurons, which are (4) Spinal Cord both its rising and falling phases, and reportedly much more sensitive to Cyclic AMP has been injected into a potentiation of the after-

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hyperpolarization. Changes in rest­ ing membrane potential and resis­ A A 20 ACP100 ACP 100 A 20 tance were less conspicuous or pre­ dictable. SO These effects, which are rather different from those observed when norepinephrine is applied outside motoneurons (Phillis et al., 1968a; Engberg et al., 1974; see Section E. II (1) (c) of this chapter) are difficult to equate with the second messenger hypothesis. The experiments em­ phasize the extraordinary difficulties facing the investigator who attempts to satisfy the criteria for establishing a second messenger function for cyc­ lic AMP. Even when the diffusional L hazards associated with the plasma Imin membrane are by-passed with an in­ A 30 AMPCP 80 AMPCPP 80 A30 tracellular injection, it is conceivable that cycli AMP may activate numer­ ous mechanisms, only one of which would be involved in the second messenger response to a specific transmitter. The sought-after effect in this instance could well be sub­ merged in a plethora of secondary phenomena.

IV. PRESYNAPTIC ACTIONS OF CYCLIC AMP There is now evidence from a number of systems that under suita­ ble conditions cyclic AMP or its

Imin dibutyryl derivative and various phosphodiesterase inhibitors can in­ AMP 20 AMPPCP40 AMPCPP 60 crease the release of synaptic trans­ mitters. A role of cyclic AMP in 40 transmitter release at the neuromus­ cular junction was first postulated by Breckenridge et al., (1967) on the basis of the observed enhancement by theophylline of the potentiation of neuromuscular transmission by epinephrine. In the rat phrenic nerve-diaphragm preparation di­ butyryl cyclic AMP and the in­ hibitors theophylline and caffeine increased the amplitude of endplate 0l_ potentials and the frequency of miniature endplate potentials (Gold­ Imin berg and Singer, 1969). These results were considered to be consistent Figure 8—A, B. Examples of lack of effect of methylene isosteres of 5'-AMP(ACP), with the hypothesis that cyclic AMP 5'-ADP (AMPCP) and the OL, /3 -methylene isostere of 5'-ATP (AMPCPP) on two plays a role in the release of acetyl­ cerebral cortical neurons which were depressed by adenosine (A). C. The j3 , choline and in the "defatiguing" ef­ y-methylene isostere of 5'-ATP (AMPPCP) had quite a pronounced depressant fect of epinephrine. Miyamoto and action on this cerebral cortical neuron, although the a , (3 -analog was inactive. Breckenridge (1974) confirmed that 5'-AMP (AMP) also depressed the neuron. The application currents in nA are given an increase in miniature endplate on the figure.

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potential frequency occurs with release of catecholamines from ad­ secretion coupling (Baker, 1972; theophylline, but were unable to ob­ renal medulla and from peripheral Miledi, 1973). serve any effect of dibutyryl cyclic and central adrenergic strucutres The lack of an appropriate tem­ AMP on miniature endplate poten­ (Berkowitzetal., 1970; Peach, 1972; poral relationship between cyclic tial frequency in normal prepara­ Wooten et al., 1973; Cubbedu et al., AMP accumulation and the secret­ tions, although it caused a transient 1975; Westfall et al., 1976). ory process may provide further increase in 15 mM K1" -stimulated While the evidence is generally grounds for objection to the in­ (fatigued) preparations. These inves­ consistent with a role for cyclic volvement of the nucleotide in tigators concluded that cyclic AMP AMP in transmitter storage and re­ excitation-secretion coupling. Thus, was probably not an obligatory fac­ lease, some questions must be raised whilst acetylcholine and nicotine in­ tor in ACh-release from non-fatigued about the interpretation of the find­ crease catecholamine release and preparations. The effects of ings. Theophylline and caffeine, the cyclic AMP formation in cat adrenal theophylline were tentatively as­ two most extensively utilized phos­ medulla, the increases are consider­ cribed to an alteration in calcium phodiesterase inhibitors, have po­ ably out of phase (Jaanus and Rubin, influx into the nerve terminal and the tent calcium mobilizing actions 1974). The possibility that the in­ positive effects of dibutyryl cyclic (Blinks et al., 1972; Serck-Hanssen, creases in cyclic AMP may be sec­ AMP observed by Goldberg and 1974; Amer and Kreighbaum, 1975). ondary to transmitter release must Singer (1969) to osmotic factors. This action could explain many of also be considered. Indeed, forma­ Miyamoto and Breckenridge (1974) the findings of enhanced transmitter tion of cyclic AMP in adrenal medul­ also failed to confirm the earlier find­ release after application of these lary tissues exposed to ing by Goldberg and Singer of an agents, as calcium entry is regarded cholinomimetics may be a result of enhancement of miniature endplate as the basic event in excitation- the action of released potential frequency in non-fatigued muscles by epinephrine. Wilson (1974), using the same preparation, SUPERIOR COLLICULUS found that dibutyryl cyclic AMP caused a significant increase in the HIPPOCAMPUS AMP CI" quantum content of the endplate po­ 60 60 60 tential and an enhancement of the releasable store of transmitter, but that it did not affect the probability of release. He suggested that cyclic AMP is involved in metabolic activ­ A C ity in the nerve terminal regulating the synthesis, mobilization and stor­ age of acetylcholine rather than in the excitation-secretion coupling process itself. CAUDATE Dibutyryl cyclic AMP can also AMP initiate activity in unstimulated CI' AMP 20 motor axons of the cat soleus nerve 10 10 10 in vivo and produce 'stimulus-bound repetitive activity' in stimulated axons (Standaert et al., 1976a, b). It appears that dibutyryl cyclic AMP can depolarize nerve terminals in B D this preparation, causing a release of .2mV transmitter and a prolongation of ac­ 2 mm 10 sec tion potentials. Dibutyryl cyclic GMP did not share this effect. Figure 9—Examples of inhibition produced by 5'-AMP in different regions of the brain. Adenosine and the calcium an­ A illustrates the histologic verification of the position of a CAi pyramidal neuron tagonist, verapamil, prevented the (arrow), and of two other neurons in the CA4 area of hippocampus. This CAi neuron effects of dibutyryl cyclic AMP was completely silenced by the microiontophoretic application of 10 nA 5'-AMP(B). which were attributed to an altera­ C illustrates the effect of 5'-AMP on the glutamate-sustained activity of a superior tion in calcium fluxes in the nerve colliculus neuron. This neuron responded to photic stimulation with a latency of terminal. 25-30 msec. In D the spontaneous activity of a caudate neuron in a decerebrate animal is readily depressed by 5'-AMP without appreciable attenuation of the spike Cyclic AMP and/or phosphodies­ amplitude. B and C are ratemeter records reproduced from the original paper terase inhibitors have also been ob­ tracing. D was recorded from an oscilloscope on slow-moving film. (From G. K. served to increase the spontaneous Kostopoulos and J. W. Phillis, 1977).

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3,5-cGMP 80 3-AMP20 GUANOSINE70 ACH 20 were quite different from those of cyclic GMP, Krnjevic and his col­ leagues concluded that the latter was unlikely to be acting as a second 50 messenger for ACh. A rather different result has been obtained with cat sensorimotor cor­ tical neurons. Both intracellularly in­ jected cyclic GMP (2 nA) and ex- tracellularly applied ACh caused in­ creases in resting membrane resis­ tance (Swartz and Woody, 1976) and the investigators concluded that cyc­ lic GMP could mediate the ACh- induced changes in membrane con­ ductance. However, in experiments in other laboratories, increases in membrane resistance have not been a consistent feature of ACh-induced depolarizations of cat cerebral corti­ 1min cal neurons (Fig. 11) or rat caudate neurons (Bernardi et al., 1976). 0l_ In conclusion, the effects of cyclic Figure 10—Ratemeter record of firing of a corticospinal neuron, Acetylcholine (ACh, GMP appear to be variable. Even in 20 nA) excited this neuron. 3',5'-cyclic GMP (80 nA) and 3'-AMP (20 nA) depressed the hands of Stone et al. (1975), the spontaneous firing. Guanosine (70 nA) was without effect. cyclic GMP inhibited nearly 20 per cent of the rat corticospinal neurons tested and excited 60 per cent. In catecholamines, perhaps acting as a bellar Purkinje cell neurons (Phillis the author's laboratory 63 per cent signal to initiate the replacement of et al., 1974; Hoffer et al., 1971). of the 50 corticospinal neurons released transmitter (Guidotti and In the belief that extracellular ap­ tested were depressed and only a Costa, 1973; Nikodijevic et al., plication may be an inappropriate small percentage were excited. Non- 1976). technique for the demonstration of a spontaneously active cortical neurons were more likely to be ex­ V. ACETYLCHOLINE AND second messenger function of cyclic CYCLIC GMP GMP, Krnjevic et al. (1976) injected cited by cyclic GMP, even though There has been much discussion this substance directly into spinal these cells are often depressed by of the possibility that cyclic GMP is motoneurons. Previously studies ACh (Phillis, 1974c). Cyclic GMP the intracellular second messenger had shown that these neurons pos­ also depresses cerebellar Purkinje for muscarinic actions of ACh sess a muscarinic cholinergic ex­ cells (Hoffer et al., 1971) which have (George etal., 1970; Lee etal., 1972; citatory receptor, which has rather been considered as chohnoceptive Goldberg et al., 1973; Kebabian et similar properties to those of cor­ (Crawford et al., 1966). The most al., 1975). This hypothesis is based ticospinal neurons (Zieglgansberger reasonable conclusion from these on the evidence that in a number of and Reiter, 1974). The most consis­ diverse findings is that cyclic GMP tissues cholinergic activity is as­ tent effect produced by intracellular may have either depressant or excit­ sociated with a rise in the tissue injections of cyclic GMP in cat ant actions and that the result ob­ content of cyclic GMP. This motoneurons was a rise in mem­ tained in any particular series of hypothesis has received direct sup­ brane conductance, acceleration in experiments will be determined by port from the finding that ACh and time course of spike potentials and various technical factors, such as the cyclic GMP have mainly excitant accentuation of post-spike hyper- choice of anaesthetic and the level of actions on rat corticospinal neurons polarizations. Alterations in mem­ spontaneous activity of the neurons (Stone et al., 1975). In a study on cat brane potential were less consis­ tested. cerebral cortical neurons cyclic tently evoked and could be either Cyclic AMP accelerated the firing GMP had either excitant or depres­ hyperpolarizing or depolarizing. of 15 per cent of cerebellar Purkinje sant actions, but these correlated Acetylcholine, applied extracellu- cells tested and it was conjectured poorly with the excitant or depres­ larly, had a depolarizing action that this resulted from pre- or post­ sant actions of acetylcholine (Phil­ which was associated with rise in synaptic effects of calcium chelation lis, 1974c). In other tests of cyclic membrane resistance, together with by the nucleotide (Siggins et al., GMP it has had depressant actions a pronounced slowing of spike re­ 1971a). If cyclic GMP, which is an on corticospinal (Fig. 10) and cere­ polarization. As the effects of ACh even less potent depressant than

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cyclic AMP, shares the same cal­ Ach 198 nA cium chelating ability, it might be iJiMiMniliinninlii.iliuilniilniJ.iiiliMiMlllLlillllinillllllllllJllHlllllllllllllllllllllllllilllllinJnill expected to have a more frequent EC excitant action than the latter. Ut+b ' p- ' 30 Sec ' JJ20 mV

VI. PHOSPHODIESTERASE RMPi 50mV INHIBITORS Considerable emphasis has been placed on the enhancement of the depressant actions of norepine­ Figure II—I.C. Intracellular potential record of a cerebral cortical neuron recorded on phrine and cyclic AMP by the phos­ a chart recorder. Acetylcholine (ACh. 198 nA) depolarized the neuron in the phodiesterase inhibitors, aminophyl­ absence of any marked alteration in membrane resistance. 0.5 nA pulses of 250 msec, duration of inward and outward current were used to test input impedance. line and papaverine (Siggins et al., E.C. Recording made after the electrode had been withdrawn from the neuron 1971; Hoffer et al., 1973). However, showing the small amount of bridge imbalance and the lack of effect of the current aminophylline and, to a lesser ex­ application on the recorded potential. (J. P. Edstrom and J. W. Phillis. unpublished tent, papaverine, have potent de­ observation). pressant actions of their own (Anderson et al., 1975; Lake et al., 1973), those of aminophylline APH30 ED 30 APH ED TPH80 ([theophylline]-2-ethylene diamine) being due to its ethylene diamine component (Fig. 12) and occurring even in animals pretreated with 50r~ 6-hydroxydopamine (Lake et al., 1973). Studies with aminophylline and papaverine have shown that ion- tophoretic applications of these drugs tend to enhance the depres­ sant actions of norepinephrine and cyclic AMP. This finding has been hailed as being consistent with the mediation of norepinephrine's ac­ tions by cyclic AMP but it might equally well be a reflection of the summative abilities of the inhibitory actions of various compounds. 0l_ In this laboratory, the phos­ phodiesterase inhibitor theophylline has been demonstrated to an­ 1 min tagonize, rather than potentiate, the depressant actions of cyclic AMP on Figure 12—Ratemeter record of spontaneous firing of a cerebral cortical neuron. corticospinal neurons, presumably Aminophylline (APH, 30 nA) and ethylene diamine (ED, 30 nA) had potent as a result of its block of the depressant effects. Theophylline (TPH, 80 nA) did not depress the neuron's firing. adenosine receptor. Finally it must be stressed that hibit the adenosine uptake Purkinje cells by interfering with phosphodiesterase inhibitors may mechanism (Mah and Daly, 1976) adenylate cyclase activation (Siggins have a variety of other actions, in­ and papaverine stimulates Na+ , et al., 1973). A related observation cluding calcium mobilization K+ -ATPase in crayfish nerves was that PGEi and PGE2 also (Blinks et al., 1972; Serck-Hanssen, (Woods and Leiberman, 1976). Such blocked the depressant action of ion- 1974; Berridge, 1975; Amer and alternative potential mechanisms of tophoretically applied aminophyl­ Kreighbaum, 1975; Moritoki et al., action must be borne in mind when line. Prostaglandin Ei failed to affect 1976), 5'-nucleotidase inhibition phosphodiesterase inhibitors are to the responses of hypothalamic (Tsuzuki and Newburgh, 1975) and be used. neurons to norepinephrine (Stitt and adenosine receptor blockage (Huang Hardy, 1975) and in a study on cere­ and Daly, 1974) by the methylxan- VII. PROSTAGLANDINS bral cortical neurons, prostaglandins thines, including isobutylmethylxan- It has been claimed that prostag­ of the E series were found to be only thine. Other inhibitors such as landins of the E series can selec­ weak antagonists of norepinephrine papaverine, diazepam, chlor-' tively antagonize the depressant ac­ inhibition but were good antagonists diazepoxide and RO 20-1724 can in­ tions of norepinephrine on cerebellar of aminophylline depression (Lake

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et al., 1973). If, as suggested above, of the membrane, and it has been experiments included lanthanum, the depressant actions of aminophyl- suggested that the amines may act in cobalt, manganese and nickel. Con­ line are due to ethylene diamine and the a -mode by increasing calcium firmation that polyvalent metallic not to phosphodiesterase inhibition, binding in the muscle membrane, ions with calcium antagonist proper­ one can only speculate about the which would somehow enhance ties can antagonize norepinephrine- relationship of this antagonism to the movements of potassium. In the evoked inhibition has been forth­ second messenger hypothesis. /3-mode, catecholamines would coming from experiments on cere­ Furthermore, the finding that pros­ immobilize membrane bound cal­ bellar Purkinje cells, in which lanth­ taglandins enhance the formation of cium or reabsorb free calcium and so anum and lead blocked cyclic AMP by cortical tissue slices prevent depolarization and the gen­ norepinephrine-evoked inhibitions (Berti et al., 1972; Kuehl et al., 1972) eration of pacemaker potentials of these neurons (Nathanson et al., lends no support to the hypothesis (Bulbring and Tomita, 1969c; 1976; Taylor et al., 1976). Lead has that they block norepinephrine by Magaribuchi and Kuriyama, 1972). been used as a calcium antagonist in interfering with cyclic AMP forma­ experiments on skeletal neuromus­ tion. Prostaglandins of the E series In the light of these observations cular junctions where, acting in a depress NE-secretion by sympathe­ and conclusions, it was considered manner comparable to lanthanum, it tic nerve terminals and there is evi­ worthwhile to observe the effects of can uncouple excitation-secretion dence that this is a result of interfer­ agents which interfere with calcium coupling in the presynaptic nerve ence with calcium availability binding on the central effects of terminals (Manalis and Cooper, (Hadhazy et al., 1976; Stjarne, 1976; biogenic amines. Given the difficul­ 1973). Similar results have been ob­ Hedqvist, 1976). ties inherent in altering extracellular tained in sympathetic ganglia where calcium concentrations in the brain, lead competitively inhibits calcium- the approach has been to use agents dependent synaptic transmission F. AMINES, CALCIUM AND with a demonstrated ability to mod­ (Kober and Cooper, 1976). NA+ , K+ -ATPase ify calcium binding to, and fluxes across, plasma membranes, as well Norepinephrine is the inhibitory Two difficulties have arisen from as calcium chelators. The effects of transmitter released by sympathetic these studies with the so-called cal­ these agents on the depressant ef­ nerve fibers onto smooth muscle cium 'antagonists'. In a study on fects of various biogenic amines on cells of the taenia coli of the large cerebellar Purkinje cells, Freedman cerebral cortical neurons were there­ intestine. This tissue has been the et al. (1975) failed to confirm an fore ascertained. The so-called cal­ subject of numerous investigations antagonist action of cobalt, man­ cium "antagonists" used in these and therefore forms a useful model ganese and verapamil on studies included various metallic ca­ of an inhibitory adrenergic synapse. norepinephrine-depressions and at­ tions, verapamil (isoptin hydroch­ The inhibitory effects of norepine­ tributed the positive findings of Yar­ loride), local anaesthetics, neomycin phrine are a result, in part, of a brough et al. (1974) to a technical and ruthenium red (Yarbrough et al., hyperpolarization due to an in­ factor — interruption of the agonist 1974; Phillis, 1974a, 1974b). The creased potassium conductance of application sequences during the chelating agents were EDTA the membrane (a -receptor activa­ period of administration of the an­ (ethylenediamine tetracetic acid) tion) and, in part, by the suppression tagonist, and a subsequent decrease and EGTA (ethyleneglyco-bis- of the pacemaker potential in the amount of agonist ejected. aminoethyl-ether-tetracetic acid). (/3 -receptor activation) (Bulbring This is unlikely to have been the With the exception of neomycin and and Tomita, 1969a and b). explanation for the positive results ruthenium red, the calcium an­ The inhibitory action of observed with cobalt, manganese tagonists tended to depress cell ex­ catecholamines on the guinea pig and verapamil for two reasons. citability during and immediately fol­ taenia coli are calcium-dependent, in Firstly, the retaining currents used lowing their application. Repeated that these amines are ineffective in a by Yarbrough et al. (1974; 8-10 nA) applications of the agonist amines calcium-free solution, whereas the were substantially lower than those were made during and after ad­ inhibitory action of epinephrine is employed by Freedman et al. (1975; ministration of the calcium an­ potentiated by an excess of calcium 15nA), and currents of this mag­ tagonists, and were continued until (Bulbring and Kuriyama, 1963; nitude would not have had such a recovery was evident (recovery Hotta and Tsukiu, 1968). Calcium pronounced effect on subsequent times were from 3-20 min). Calcium antagonists such as manganese and amine ejections. Secondly, after antagonists antagonized the depres­ ruthenium red abolish the action of completion of the antagonist applica­ sant effects of norepinephrine, the catecholamines on smooth mus­ tion, the amines were applied in a dopamine, serotonin, histamine and cle cells of the taenia coli even in the repeated sequence until recovery acetylcholine on cerebral cortical presence of calcium (Bulbring and occurred. During this period an­ neurons, but did not alter inhibitions Tomita, 1969c; Tomiyama et al., tagonism was often pronounced for evoked by 7 -aminobutyric acid, or 1973). Calcium produces a similar several minutes and then gradually acetylcholine excitations. effect to the catecholamines, causing disappeared. The positive results a hyperpolarization and stabilization The metallic cations tested in our obtained by others with lead and

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lanthanum, referred to above, sup­ their experiments in the extracellular their action. To a large extent, the port our conclusion that polyvalent environment. It is impossible to use of these agents in physiological metallic cations can antagonize conclude therefore that the research was pioneered in studies on amine-evoked depressions. Furth­ phenothiazines were not acting as calcium fluxes in cardiac muscle, ermore, two calcium 'antagonists' calcium antagonists in these experi­ and doubts are now being voiced which do not depress neuronal ex­ ments. Further, no attempt was about their pharmacological specific­ citability, neomycin and ruthenium made to ascertain whether the an­ ity in this system (Kass and Tsien, red, also block amine-induced de­ tagonism exerted by the 1975). It is becoming increasingly pressions (Phillis, 1974b). It should phenothiazine derivatives could dis­ apparent that many calcium an­ be stressed that in these latter exper­ criminate between different amines tagonists also affect two enzymes iments there was no interruption of rather than between norepinephrine that may be of crucial importance in the agonist ejection sequence. A and two very different agents, the regulation of membrane exctia- more likely explanation for the fail­ 7-aminobutyric acid and cyclic bility, Na+ , K+ -ATPase and ure of Freedman et al, (1975) to AMP. adenylate cyclase. The metallic ca­ replicate our results lies in the 'cal­ A characteristic finding in our tions, lithium, lead, copper, zinc, cium antagonist' potencies of the studies with the calcium 'an­ iron, cobalt, calcium, nickel, man­ polyvalent metallic cations. When tagonists' has been a simultaneous ganese and mercury inhibit brain tested as inhibitors of the calcium- block of the actions of histamine, Na+ , K+ -ATPase (Skou, 1965; dependent spike potential of barna­ serotonin and norepinephrine, im­ Donaldson et al., 1971; Ting-Beall et cle muscle fibers, cobalt and man­ plicating a common mode of depres­ al., 1973; Specht and Robinson, ganese had approximately 1/1 Oth sion by the various amines. This 1973; Prakash et al., 1973; Hexum, and l/50th the activity of lanthanum apparent lack of pharmacological 1974). Lanthanum inhibits Na+, (Hagiwara, 1973). It is hardly sur­ specificity in the action of the K+ -ATPase from heart muscle prising therefore that positive results amines on spinal cord motoneurons (Nayler and Harris, 1976), and can more readily be obtained with has prompted Engberg and his col­ lanthanum, manganese and lanthanum than with cobalt and leagues to propose a common ruthenium red inhibit Mgt"t , manganese. mechanism of action for all these Ca ++-ATPase activity of rat cor­ compounds, mediated perhaps on tex synaptic membranes (Ichida et The antagonism by phenothiazine charged groups at the membrane al., 1976). Other agents which have derivatives (fluphenazine, flupen- (Engberg et al., 1974). been used as calcium antagonists thixol and chlorpromazine) of + The recent discovery of specific and which also inhibit Na , norepinephrine-evoked depression antagonists which will discriminate K+-ATPase include alcohols and of cerebellar neurons has been inter­ between the actions of histamine and local anaesthetics (Seeman, 1972). preted as suggesting that calcium is those of the other amines not involved in the action of this (metiamide; Phillis et al., 1975), and Brain adenylate cyclase is inhi­ amine, since the antagonism by between those of serotonin and the bited by low concentrations of chlorpromazine of norepinephrine's other amines (metergoline; Sastry lithium, lead, copper, zinc, lanth­ effects in vitro occurs at concentra­ and Phillis, 1977a) on cerebral corti­ anum and mercury (Forn and Val- tions some two orders of magnitude cal neurons strongly supports the decasas, 1971; Taylor et al., 1976; less than those required to block concept of individualized receptors Nathanson and Bloom, 1976; calcium fluxes (Freedman and Hof- for each of the major groups of Nathanson, Freedman and Hoffer, fer, 1975). Thus while chlor­ amines, each receptor being con­ 1976; Walton and Baldessarini, promazine blocks the stimulation of nected to a similar or common 1976). Cobalt inhibits cardiac sar- adenylate cyclase by dopamine at a mechanism which, when activated, colemmal adenylate cyclase in the concentration of 10~6 M (Miller et evokes a membrane hyperpolariza- presence of magnesium (St. Louis al., 1974), a concentration of 5 x 10~4 tion. It remains to be clearly estab­ and Sulakhe, 1976), and manganese is required to block calcium uptake lished whether, when applied ion- reduces the stimulation of rat brain into synaptic plasma membrane re­ tophoretically, the phenothiazines enzyme by dopamine (Walton and sidues (Madiera and Antunes- can discriminate between Baldessarini, 1976). Antagonism of Madiera, 1973). catecholamines and the other norepinephrine-evoked depression amines. lontophoretically applied of hippocampal or cerebellar Several objections can be raised to neurons by lithium, lanthanum and this somewhat specious interpreta­ chlorpromazine antagonizes the de­ pressant action of histamine on lead has been attributed to inhibition tion of their findings by Freedman of adenylate cyclase activity (Segal, and Hoffer (1975). Quite apart from cerebral cortical neurons (Phillis et al., 1968b), and this substance will 1974; Nathanson et al., 1976; Taylor the difficulties inherent in compari­ et al., 1976). sons between in vivo and in vitro likely have to be discarded as a selective catecholamine antagonist. observations on a variety of prepara­ It is difficult to escape the conclu­ tions, the authors can have had no A more significant problem as­ sion that agents such as the polyval­ knowledge of the actual concentra­ sociated with the use of calcium an­ ent cations described above, which tion of the phenothiazines attained in tagonists may lie in the specificity of possess the ability to antagonize cal-

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cium binding and fluxes in the mem­ free lithium solution. Extracellular formed causes the synthesis and/or brane, will often also inhibit Naf , potassium is necessary for the + release of an intracellular agent K -ATPase and adenylate cyclase. generation of a maximal hyper­ which acts directly on Na+ , The latter property may reflect the polarization by serotonin (Watanabe K+ -ATPase, enhancing its activity ability of the calcium antagonists to and Koketsu, 1973; Koketsu and (Rogus et al., 1977). bind to negatively charged mem­ Shirasawa, 1974). Na+ , K+ -ATPase activity in brane sites, including the metal- Comparable responses have been mammalian brain homogenates or binding regulatory sites associated recorded from avian and amphibian synaptosomal fractions is stimulated with enzymic systems, displacing skeletal muscles, which are hyper- by various amines, including other metallic ions. These agents polarized by catecholamines by a norepinephrine, dopamine, seroto­ may thus interfere with a number of ouabain and potassium sensitive nin, histamine and acetylcholine processes which do not normally re­ process (Somlyo and Somlyo, 1969; (Schaefer et al., 1972; Yoshimura, quire calcium as a co-factor. For this Bressler et al., 1975; Koketsu and 1973; Godfraind et al., 1974; Gilbert reason extreme caution will obvi­ Ohta, 1976). Frog skeletal muscle et al., 1975; Logan and Donovan, ously be necessary for the interpre­ membrane contains a sodium pump 1976; Sulakhe et al., 1976; Desaiah tation of results obtained in studies which is stimulated by epinephrine, and Ho, 1976; Kometiani et al., which rely solely on the use of cal­ greatly increasing sodium efflux. 1976; S. L. Lee and J. W. Phillis, cium antagonists. Similar considera­ This action of epinephrine is blocked unpublished observations). Stimula­ tions must apply to the use of chelat­ by strophanthidin (Hays et al., tion by norepinephrine was an­ ing agents, which can bind other 1974). Further studies on this tagonized by the a -adrenore- metallic cations in addition to cal­ phenomenon have revealed that ceptor blocker, phenotolamine cium (Garvan, 1964). Nevertheless, Na+ , K+ -ATPase activity in the (Iwangoff et al., 1974; Gilbert the observation that EDTA and sarcolemmal fraction from rat et al., 1975). Acetylcholine may EGTA can abolish the depressant skeletal muscle is stimulated by also inhibit the enzyme (Gilbert et action of norepinephrine on cerebral various catechols and their or- al., 1975; Kometiani et al., 1976). cortical neurons (Phillis, 1976) is thoquinones, including D-, Cyclic AMP reportedly does not consistent with the assumption that L-isoproterenol and D-, L-epine- alter Na+ , K+ -ATPase activity in the presence of calcium or a related phrine and pyrocatechol, all of brain preparations (Yoshimura, metallic cation is necessary for which have comparable activity. 1973; Sulakhe et al., 1976), indicat­ norepinephrine to exert its effects. Phenylephrine, an a -adrenergic ing that the stimulant effect of the The possibility that the slow in­ agonist, was unable to enhance biogenic amines is probably not a hibitory postsynaptic potential of Na+ , K+ -ATPase activity, as were direct consequence of activation of sympathetic ganglion cells, in the cyclic AMP and dibutyryl cyclic adenylate cyclase. generation of which catecholamines AMP (Cheng et al., 1977). Within the central nervous system have been implicated, is evoked by j3 -adrenergic blocking agents, prop­ Na+ , K+ -ATPase is known to be activation of an electrogenic sodium ranolol and dichloroisoproterenol, associated with synaptic mem­ pump was discussed in Section had no effect on the enhancement of + + branes. Hosie (1965) and Kurokawa B.I.(2). Na-*- > K+ -ATPase in­ Na , K -ATPase activity pro­ et al. (1965) found Na+, hibitors such as ouabain, sodium duced by L-isoproterenol, suggest­ K+ -ATPase of high specific activity cyanide, 2,4-dinitrophenol and low ing that the effect was not mediated in subcellular fractions containing temperature selectively block the through classical /3 -receptors. In isolated nerve endings derived from slow IPSP and the hyperpolarizing contrast, when the same inves­ cerebral tissues, and this enzyme action of epinephrine on bullfrog tigators (Rogus et al., 1977) studied represented a substantial portion of ganglion cells, presumably by inter­ changes in sarcoplasmic Na+ and + the activity of the total brain ference with the functioning of the K content in intact muscles ex­ homogenate. The enzyme in rat electrogenic pump. Perfusion with posed to L-isoproterenol, they found cerebral cortex has been localized sodium-free lithium or hydrazinium evidence for a /3 -adrenergic stimula­ 1- histochemically in neuronal proces­ solutions, which might be expected tion of the membrane Na+ , K" ses, including axons and dendrites to reduce the intracellular sodium pump. D-isoproterenol had only 3% (Stahl and Broderson, 1976). concentrations, abolish the slow of the activity of the L-isomer, and Pharmacological studies on rat IPSP and catecholamine-induced the response to L-isoproterenol was cerebral cortical neurons support the hyperpolarization (Nishi and blocked by propranolol, but not by hypothesis that amines depress by Koketsu, 1968; Koketsu, Shoji and phentolamine. The effect of activating Na+ , K+ -ATPase. Thus Nishi, 1973; Koketsu and Nakam- L-isoproterenol on ionic content ouabain and lithium antagonize the ura, 1976). The hyperpolarizing ac­ could be mimicked by dibutyryl cyc­ depressant actions of norepine­ tion of serotonin on nicotinized lic AMP or caffeine. The results of phrine on these neurons (Phillis, bullfrog ganglion cells may also be the second study have led to the 1974b, 1976; Phillis and Limacher, mediated by an electrogenic pump, suggestion that in intact muscle 1974), and it has recently been re­ as it is blocked by ouabain, or ex­ /3-adrenergic agonists activate ported that ouabain antagonizes the posure of the ganglion to sodium- adenylate cyclase. Cyclic AMP thus depressant actions of norepine-

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phrine and dopamine on cerebellar Purkinje cells and caudate neurons respectively (Yarbrough, 1976). Further support for the hypothesis has been sought by testing the ef­ fects of a diverse series of Na + , DIGITOXIN 15 K+-ATPase inhibitors including various glycosides, aglycones, NA60 NA NA NA NA ethacrynic acid and harmaline (Table 3) on depressions of cortical neurons evoked by the biogenic 60 amines (Figs. 13, 14; Sastry and Phil- lis, 1977b). A close correlation was observed between the known poten­ cies of the compounds tested as Na + , K+-ATPase inhibitors and their ability to completely or par­ tially antagonize amine-induced de­ STROPHANTHIN K 15 pressions. The inhibitors did not an­ 5-HT tagonize the depressant effects of 5-HT 80 5-HT 5-HT calcium, y -aminobutyric acid and AMP, implying that these agents are unlikely to exert their effects through activation of an electrogenic sodium pump. Other agents which have been re­ ported to inhibit Na+ , K.+ -ATPase (Roufogalis and Belleau, 1969; Is­ rael, 1970; Seeman, 1972), and which also antagonize the depres­ sant actions of the biogenic amines 2min on central neurons include the bar­ biturate anaesthetics, ethanol, and the catecholamine antagonists, chlorpromazine, haloperidol, di- Figure 13— The antagonism of norepinephrine (A, NA, 60 nA) and 5-hydroxytryptamine (B, 5-HT, 80 nA) effects by digitoxin (15 nA) and strophanthin benamine and phenoxybenzamine K (15 nA) respectively. A and B are records from different deep spontaneously firing (Phillis, 1976). cerebral cortical neurons. In this and the subsequent figures the ordinate calibration Of considerable interest to the represents neuronal firing rate as spikes per second. The horizontal bars indicate the present discussion is the report that duration of drug application periods. (From B. S. R. Sastry and J. W. Phillis. 1977b).

TABLE 3 Effects of Net+, K + -A TPase Inhibitors on the Depression of Cerebral Cortical Neurones by the Biogenic Amines and Other Agents

Noradrenaline Histamine 5-HT GABA Adenosine S'AMP Calcium

Ethacrynate 31/36* 25/31 17/21 0/5 0/9 0/5 0/6 Ouabain 17/22 23/27 16/19 0/7 0/5 0/5 0/5 Harmaline 18/19 23/25 19/20 0/5 0/8 0/5 0/5 Digitoxin 7/8 6/6 4/4 0/7 Digitoxigenin 5/6 4/5 3/4 0/5 Strophanthin K 5/5 5/5 5/5 Strophanthidin 4/6 4/5 4/4

"The denominator indicates the total number of cells tested and the numerator shows the number of cells in which the effect of an agonist is antagonized. (Data from Sastry and Phillis, 1977b).

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norepinephrine and cyclic AMP- The evidence presented in this any alteration in (or in some in­ induced hyperpolarizations of rat section suggests that transmembrane stances with a decrease in) mem­ pineal gland cells are blocked by alterations in calcium levels may be brane conductance. ouabain or elevated potassium (Par- crucial for the mediation of the ac­ The role of cyclic nucleotides in fitt et al., 1975). The investigators tions of the biogenic amines. Cal­ the control of calcium binding and concluded that norepinephrine ex­ cium has multiple functions within calcium fluxes is presently uncer­ erts its hyperpolarizing effects by innervated tissues, including the tain. Recent reports indicate that activation of adenylate cyclase, al­ maintenance of membrane integrity, cyclic AMP can initiate calcium se­ though they could not exclude the excitation-secretion coupling, questration by endoplasmic re­ possibility that the hyperpolarization excitation-contraction coupling and ticulum of skeletal, cardiac and and the increase in cyclic AMP con­ regulation of enzymic activity (Bian- smooth muscle through stimulation tent of the gland occurred indepen­ chi, 1968; Triggle, 1971). An in­ of a calcium pump (Berridge, 1975). dently. The most significant obser­ volvement in the generation of post­ Mitochondria are also capable of se­ questering calcium against a consid­ erable concentration gradient ETHACRYNATE 10 (Drahota et al., 1965) and may play a HA 80 Ca2*30 HA Ca' HA Ca' considerable role in regulating in­ tracellular calcium levels (Borle, 1973). There is abundant evidence that cyclic AMP can also release calcium from intramembranous or intracellu­ lar stores, possibly including the mitochondria, in presynaptic termi­ nals and secretory cells (Rasmussen,

OUABAIN 10 1970; Rasmussen et al., 1972; Ber­ ridge, 1975) (see section E, IV). For 2* HA Ca' HA Ca.2' * HA Ca*' instance, the stimulatory effect of B cyclic nucleotides on epinephrine 60 secretion from the adrenal medulla persists in calcium-free conditions which completely block the stimulatory effects of both high potassium and nicotine (Peach, 1972). 0L The methylxanthine, caffeine, can stimulate a release of calcium from 4min adrenal medullary mitochondria (Rahwan et al., 1973) and this may Figure 14— The antagonism of histamine (HA, 80 nA) — induced depression of a account for the secretion enhancing spontaneously active cerebral cortical neuron by ethacrynate (A, 10 nA) and ouabain (B, 10 nA). Both HA and calcium (Ca+ + , 30 nA) were applied in sequence properties of this group of phos­ at regular intervals. Note that ethacrynate and ouabain more effectively prevented phodiesterase inhibitors. Calcium re­ the late prolonged phase of the amine-induced inhibition and did not antagonize the lease from intramembranous or in- depression by Ca + +. A-B depicts a continuous recording of a deep cortical cell tracytoplasmic stores by cyclic AMP firing (From B. S. R. Sastry and J." W. Phillis, 1977b). may occur in both presynaptic and post-synaptic neuronal elements and could account for some of the effects vation, however, was that the cyclic synaptic potentials must now be of extra- or intra-cellularly applied AMP-induced hyperpolarization was considered for inclusion with these cyclic AMP and dibutyryl cyclic generated by a ouabain sensitive actions. Elevation of intraneuronal AMP. ionic mechanism, presumably re­ calcium levels causes a membrane Calcium-cyclic nucleotide interac­ flecting activation of Na+ , hyperpolarization which is thought tions will occur in several other re­ K+-ATPase. Reversal of the to be the result of an increase in spects. Calcium appears to be in­ norepinephrine-evoked hyperpolari­ membrane potassium conductance volved in regulation of adenylate and zation of frog skeletal muscle by (Krnjevic and Lisiewicz, 1972). This guanylate cyclase, and alterations in elevated potassium has been re­ increase in membrane conductance calcium levels may alter the re­ ported (Bressler et al., 1975) and was precludes any suggestion that sponses of these enzymes to synap­ interpreted as evidence for some amines hyperpolarize simply by in­ tic transmitters. Calcium ions can flexibility in the Na+ -K.+ transport creasing intracellular calcium, since regulate the activities of phos­ ratio of the pump. their action occurs in the absence of phodiesterases, and changes in cal-

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cium concentration within the action is consistent with the obser­ brain bundle, through which biological range can reverse the ef­ vation that the Na+ , K+ -ATPase norepinephrine-containing fibers as­ fects of cyclic AMP on some phos­ inhibitor, ouabain (Godfraind et al . cend to the cerebral cortex, is ap­ photransferase reactions (Ueda et 1971), and barium ions (Krnjevic et parently of considerable significance al., 1973). al., 1971b) excite corticospinal in the learning process (Crow, 1973). If both adenylate cyclase and cal­ neurons. The action of barium in Destruction in rats of the locus cium are involved in the response to particular is very similar to that of coeruleus from which these neurons catecholamines, the question be­ acetylcholine in that it excites arise results in a deficit in the acquis­ comes one of whether the initial neurons which are depolarized by ition of runway behavior (Anlezark event is an alteration in calcium acetylcholine and depresses neurons et al., 1973). Furthermore, central fluxes or binding which could then which are either unaffected or inhi­ administration of norepinephrine to affect the activities of cyclase, phos­ bited by acetylcholine (Krnjevic et rats previously depleted of the photransferases and phosphodies­ al., 1971b; Phillis and Limacher, catecholamine by administration of terases, or an activation of adenylate 1974). Experiments on sodium ion the dopamine- (3 -hydroxylase in­ cyclase and subsequent alteration in transport in isolated toad bladder hibitor diethyldithiocarbamate re­ intracellular or intramembranous have demonstrated that barium in­ sults in an enhancement of memory calcium concentrations. Indeed, the hibits sodium ion transport by an (Stein et al., 1975). two series of events may occur action on an electrogenic sodium Some indication of the manner in simultaneously rather than sequen­ pump (Ramsay et al., 1976). Acetyl­ which catecholamines are involved tially (Berridge, 1975). choline inhibition of cerebral cortical in short term memory may be evi­ A final step in the amine-induced neurons is prevented by various cal­ dent in the observations of Libet et hyperpolarization appears to be ac­ cium 'antagonists' (Yarbrough et al., al. (1975) on rabbit superior cervical tivation of membrane Na+ , 1974) and may result from stimula­ ganglion. Dopamine can produce a + K+-ATPase. Several tentative exp­ tion of Na+ , K -ATPase (Kome­ specific and enduring enhancement, lanations can be provided for this tiani et al., 1976). lasting at least several hours, of the event, including cyclic AMP or cal­ subsequent responses to acetyl­ cium stimulated phosphorylation of choline or the muscarinic agonist, the ATPase (Dowd and Schwartz, G. CYCLIC NUCLEOTIDES acetyl- /? -methylcholine. The 1975; Dowd et al., 1976; Sulakhe and AND MEMORY dopamine-induced enhancement of St. Louis, 1976) with an increase in The possibility that alterations in acetyl- (3 -methylcholine-depolariza- its activity; release or stimulation of the phosphorylation state of synap­ tions is mimicked by dibutyryl cyclic an ATPase-activating factor by cyc­ tic proteins might be associated with AMP applications, and the enhance­ lic AMP (Rogus et al., 1977) or cal­ short term memory was proposed by ment produced by cyclic AMP oc­ cium; or, since calcium exerts an Greengard and Kuo (1970). It was cludes any further enhancement by inhibitory action on ATPase, re­ envisaged that transmitter-evoked subsequent doses of dopamine. Ex­ moval of calcium by a cyclic AMP- increases in cyclic AMP might cause posure of the ganglion to dibutyryl or calcium-stimulated sequestration alteration in the degree of phos­ cyclic GMP within 4 min. of the do­ system. phorylation of membrane proteins pamine or cyclic AMP injection dis­ It has been suggested that and could thus lead to a short term rupts the expected modulatory ef­ acetylcholine-evoked depolarization modification of the properties of the fect. When treatment with cyclic of corticospinal neurons results from postsynaptic neuronal membrane. In GMP was delayed for periods of a decrease in membrane potassium addition to changes in the phos­ longer than 4 min. after dopamine, conductance (Krnjevic et al., 1971a), phorylation of proteins in the mem­ progressively lesser disruptions oc­ resulting perhaps from a reduction in brane, short term modulation of curred. With delays of greater than internal free calcium (Krnjevic et al., synaptic transmission could also 10-15 min. there was no disruption. 1975). However, in other experi­ occur by phosphorylation processes These features of the actions of cyc­ ments (Fig. 11, and Bernardi et al., involving enzyme induction lic GMP suggest that after the initial 1976) this decrease in membrane (Guidotti et al., 1975) or regulation modulatory change is induced by conductance has not been a promi­ of enzymes involved in the dopamine, through a cyclic AMP nent feature of acetylcholine-evoked metabolic functions of the cell. mediated mechanism, a 'storage' depolarizations. An alternative ex­ Norepinephrine in the forebrain is process develops which leads to a planation for the depolarization may thought to play a critical role in the more durable form of modulatory be that acetylcholine, directly or in­ associative learning process, and change. The storage process, but not + directly, inhibits the Na+ , K - drugs which affect norepinephrine the durable product, can be dis­ ATPase activity of corticospinal metabolism or release can affect the rupted by cyclic GMP. neurons. An inhibitory action of memory consolidation process + This dopamine-modulating action acetylcholine on brain Na+ , K - (Kety, 1972). Only two examples of in the sympathetic ganglion may ATPase has been reported (Gilbert the numerous experiments on provide an interesting model in et al., 1975; Kometiani et al., 1976), norepinephrine and learning will be which the development of short and and this suggested mechanism of mentioned here. The medial fore- long term memory traces can be

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studied. Such amine-modulating ac­ block of adenosine uptake, have L-glutamate is thought to have a tions may not be unique to the cen­ been mentioned on several occa­ predominantly postsynaptic locus of tral nervous system. Adrenaline en­ sions in this paper and could account action, and suppression of hances the sensitivity of (3 -cells in for many of the results that have L-glutamate-evoked firing of an the pancreas to subsequent doses of been reported. otherwise quiescent cell should be acetylcholine by a calcium- considered as indicative of a depres­ Regardless of the role, or lack dependent process (Burr et al., sant action of a neurotransmitter on thereof, of cyclic nucleotides in the 1976), a phenomenon which may be the postsynaptic neuron. Con­ synaptic transmission process, there similar to that described above for versely, a reduction of spontaneous is pharmacological evidence for an dopamine in sympathetic ganglia. firing, with less of an effect on involvement of calcium and Na+ , glutamate-evoked discharges, would K+-ATPase in the responses of be indicative of a presynaptic locus central neurons to the various H. CONCLUSIONS of action. It can be anticipated that biogenic amines. Biochemical On the basis of the information many putative neurotransmitters studies on the relationships between presented in this paper, it is dif­ will have actions on both pre- and amines, calcium and Na+ , ficult to reach any firm decision re­ post-synaptic membranes and that K+ -ATPase are as yet in their in­ garding the role of cyclic AMP (or the postsynaptically recorded event fancy and even the nature of the cyclic GMP) in synaptic transmis­ will reflect the outcome of actions at amine-receptors is but poorly de­ sion in the brain. While it is clear both sites. fined. Further experimentation may that cyclic AMP levels can be al­ well yield convincing evidence for a The use of glutamate-evoked fir­ tered by the exposure of neural tis­ functional interrelationship between ing in neuropharmacological exper­ sues to various neurotransmitters, it cyclic AMP, calcium and Na+ , iments has been criticized, as has would be premature to claim that K+ -ATPase. At the moment, there­ experimentation on non- this nucleotide is, or is not, essential fore, the most judicious attitude is physiologically identified neurons to the transmission process in the undoubtedly one which accepts the (Bloom, 1974). Both criticisms are pre- or post-synaptic components of likelihood of an involvement of both clearly specious and can be refuted, the synapse. In future experiments, calcium and cyclic AMP in the even though it is obviously desira­ it will be necessary to consider more mediation of amine-induced inhibi­ ble, from the point of view of repro­ critically whether extracellularly tions. ducibility, to use identifiable cells applied cyclic AMP merely provides whenever possible. To use a by now a source of adenosine and is thus A frequent but perplexing obser­ familiar example, within the cerebral activating an extracellularly located vation has been that of an increase in cortex only the corticospinal adenosine receptor, or whether it is neuronal membrane resistance dur­ neurons, which constitute a small actually reaching the hypothetical ing the application of amines or minority of the total cell population, sites at which it might act as a sec­ acetylcholine. It has generally been can be definitively identified by in­ ond messenger. The application of assumed that these were a result of vasion of an antidromically prop­ cyclic AMP by intracellular injection direct transmitter-induced altera­ agating action potential. The major­ techniques should minimize this par­ tions in postsynaptic membrane ity of the cerebral cortical neurons ticular problem, although possibly at conductances and in many instances cannot be identified in this manner, the expense of new difficulties. Prior mechanisms of action of the trans­ and to suggest that their existence blockage of the adenosine receptor mitter have been predicated on this should be ignored is clearly an un­ with agents such as theophylline or alteration in membrane resistance. realistic proposition. Unlike the cor­ adenine xylofuranoside may also as­ As we have seen (Section B. I. 3), ticospinal neurons which are charac­ sist in the categorization of re­ both acetylcholine and the cate­ terized by their spontaneous activ­ sponses to extracellularly applied cholamines can depress trans­ ity, many of the more superficial cyclic AMP as being a result either mitter release from presynaptic ter­ neurons are quiescent and can only of activation of the adenosine recep­ minals and this must therefore be be tested with extracellular applica­ tor or of some other mechanism. considered as an alternative poten­ tions of an excitant, such as glutamic Ultimately, the development of tial mechanism of action. A reduc­ acid. Even though the use of gluta- highly specific inhibitors for adeny­ tion in the release of excitatory mate may introduce another factor late cyclase should provide a firm transmitter could be associated with into the experimental situation, it basis from which to draw conclu­ an increase in membrane resistance, appears to be clearly more desirable sions about the role of cyclic AMP in and this may account for such in­ than the alternative of ignoring the synaptic transmission. creases in membrane resistance existence of these neurons. Finally, The use of phosphodiesterase in­ when they have been observed. The as indicated above, the use of excit­ hibitors in studies on cyclic nuc­ use of pulses of excitant dicarbox- ant amino acids may provide a uni­ leotides must also be approached ylic amino acids, such as que technique for separating pre- with caution. The diverse actions of L-glutamate, may provide a valuable and post-synaptic actions of ion- many of these compounds, which technique for distinguishing between tophoretically applied pharmacolog­ include calcium mobilization and pre- and post-synaptic actions. ical agents. Thus an early indication

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