Messengermoleculesin the cerebellum C h r i s t o p h e r A. Ross, D a v i d Bredt and S o l o m o n H. Snyder
ChristopherA. Ross, DavidBredtand SolomonH. Snyder are at the Departmentsof Neuroscience, Pharmacologyand MolecularSciences, Psychiatryand BehavioralSciences, TheJohnsHopkins UniversitySchoolof Medicine, 725 North Wolfe Street, Baltimore,MD 21205, USA.
As data accumulate, the mammalian brain reveals its complex and subtle synaptic mechanisms. In the simplest system, a neurotransmitter binds to the receptor portion of a molecular complex incorporating an ion channel and thus alters the membrane potential, leading to excitatory or inhibitory effects. In more complex systems, receptors are coupled to second messenger systems to generate signals of longer duration and to modulate more diverse molecular mechanisms. The cerebellar cortex has a relatively simple wiring diagram with the primary neurotransmitter of most inhibitory and excitatory synapses well established. The second messenger signalling systems are more complex and those of the cerebellar output, the Purkinje cells, are the best characterized. More recently, molecules that might act as neuromodulators, carrying messages between neurons and between neurons and glial cells, have been identified, such as endothelin and nitric oxide. The classic neurotransmitters and novel neuromodulators, together with second messenger-activated trophic factors, can interact in complex ways; in this review Christopher Ross, David Bredt and Solomon Snyder discuss how studies of cerebellar circuitry and biochemistry are revealing such interrelations. The synaptic diversity that permits complex information transfer in the brain has often been ascribed to its hundred or more putative neurotransmitters. Because only a handful of second messenger systems exist, they have not been a major focus for the exploration of synaptic subtleties. However, recent demonstrations of marked molecular heterogeneity and regional specificities within the cAMP and phosphoinositide (PI) systems provide rich possibilities for signal discrimination. Novel 'intercellular' messengers, such as nitric oxide and adenosine, afford unprecedented ways for regulating neuronal communication. The cerebellum is an ideal site for investigating these 'messenger molecules'. Thus, elements of the PI cycle, such as the inositol 1,4, 5-trisphosphate (IPz) receptor, are enriched several hundred-fold in the cerebellum compared with other brain regions or peripheral tissues. Moreover, neuronal connectivity in the cerebellum is surprisingly straightforward. Accordingly, we focus on the cerebellum to review recent advances in biochemical messenger systems in brain function.
Cerebellar wiring diagram and neurotransmitters The relative simplicity of the wiring diagram of the cerebellar cortex has made it a favored site for analysing specific synaptic interactions 1,2. The cerebellar cortex has only three major neuronal inputs and a single neuronal output (Fig. 1). One input is via mossy fibers, with cell bodies located in the spinal cord, the pontine nuclei, and other sites, and terCorrespondence should be addressedto Solomon H. Snyder. 216
© 1990, ElsevierSciencePublishersLtd,(UK)
minals on specialized granule cell dendritic complexes called cerebellar glomeruli. Granule cells then contact dendrites of the Purkinje cells via excitatory parallel fibers, which release glutamate. The second input is via climbing fibers, whose cell bodies are mostly in the inferior olive of the brain stem and whose axons terminate directly on individual Purkinje cells. A third, modulatory input arises from monoaminergic neurons in the locus coeruleus and raphe nuclei, with terminals predominantly on Purkinje cells. The output of the cerebellum derives from Purkinje cells, whose axons project to the brain stem and deep cerebellar nuclei. The two main cerebellar inputs, climbing fibers and mossy fibers, are excitatory, while Purkinje cells, which contain GABA, are inhibitory. There are three major interneurons, all capable of synthesizing GABA3 and thus inhibitory, in the cerebellar cortex - basket cells, stellate cells and Golgi type II cells. Basket and stellate cells, whose perikarya lie in the molecular layer, receive afferents from parallel fibers and inhibit Purkinje cells. Golgi cells, whose perikarya are in the granule cell layer, receive impulses from parallel fibers, as well as direct connections from mossy fibers. Their output, also inhibitory, feeds back to the cerebellar glomeruli, and thus influences the transfer of information from the mossy fibers to granule cells. Presumably, GABA released from stellate, basket and Golgi II cells directly opens chloride ion channels via GABA receptors. Autoradiographic studies show GABAA receptors to be most concentrated in the granule cell layer, and GABAB receptors in the molecular layer 4,5. In addition, the 72 subunit of the GABAA receptor, important for benzodiazepine binding, is expressed by Purkinje cells6. As the sole neuronal output of the cerebellum, the Purkinje cell is the final common path of cerebellar activity. It is also uniquely enriched in elements of the PI cycle, which may relate to certain of its properties. For instance, a single Purkinje cell receives as many as 200 000 synaptic junctions, the most of any central neuron. These inputs come predominantly from parallel fibers of granule cells, which employ a PIlinked glutamate receptor (see below). Additionally, Purkinje cell dendrites display prominent action potentials dependent on calcium (Ca2+). Granule cells comprise some 90% of all cerebellar neurons and are the only intrinsic neurons with an excitatory transmitter 1,2. Their parallel fibers synapse exclusively upon the dendritic spines of Purkinje cells, each one innervating the distal dendrites of many Purkinje cells. Because of the great abundance of granule cells, the parallel fiber-Purkinje cell synapse is quantitatively the predominant synapse in the cerebeilar cortex. By contrast, each climbing fiber invests the soma and proximal dendrites of a single Purkinje cell, like a vine enveloping a tree; climbing fibers powerfully excite Purkinje cells, with Ca 2+dependent plateau potentials and multiple spikes 7,s. The actions of excitatory transmitters in the cerebellum appear to be complex. Multiple lines of
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Fig. 1. Neurotransmission in the cerebellar cortex. (A) Simpfified wiring diagram and neurotransmitters. Basket and stellate cells are distinct but, for simplicity's sake, are indicated as interchangeable as they have similar inputs and outputs and use the same transmitter. (B) 5elected receptors and second messenger systems discussed in the text. For visual simpficity, the basket cell is not shown in (B). It synthesizes NO, which presumably diffuses to the Purkinje cell to activate cGA4P formation. In (A) and (B) dark red and light red structures are inhibitory. Black and grey structures are excitatory (except the glial cell, which is indeterminate). Abbreviations: GABA, 7-aminobutyric acid; 5-HT, serotonin; NE, noradrenaflne; Quis-PI, quisqualate PI-stimulating subtype of glutamate receptor.
Parallel fiber (glutamate)
A
Basket or stellate cell (GABA)
Purkinje cell (GABA)
Granule cell (glutamate)
- " •Glial cell
Climbing fiber (excitatory amino acid) input from • medulla J
evidence indicate that glutamate Purkinje cell axon (GABA) is the transmitter of parallel output to deep fibers9-11. Fewer data are available cerebellar nuclei for climbing fibers, whose transmitter is generally thought to be aspartate or glutamate 12, though homocysteate has also been proposed 1:~. Thus far, synaptic responses at aspartate receptors have not been clearly differentiated from B Adenosine glutamate receptors. The primary receptor subtypes of glutamate receptors in the brain directly influence ion channels and are classified according Adenosine formation to the glutamate derivative that is most selective H. The subtypes are designated N-methyl-D-aspartate (NMDA; the most effective), kainate, and quisqualate. Recently, glutamate receptors that act through the PI second messenger system to mobilize intracellular cGMP Ca~+ have been identified15-17. The formation glutamate receptor subtype that stimulates PI turnover differs in pharmacological specificity from any Excitatory of the known ionotropic receptors, amino acid being most sensitive to quisrelease qualate, but insensitive to the ionotropic agonist AMPA (o~-amino-3hydroxy-5-methyl-isoazole-4-proprionate), and the antagonist CNQX (6-cyano-2, 3-dihydroxy-7nitroqukloxaline) 15-17. Intraeellular mediators
Golgi cell (GABA)
Monoaminergic fiber (NE or 5-HT) input from locus coeruleus or raphe Mossy fiber input from pons and / spinal cord
Glutamate release
See Fig. 2
~
QUIS-PI receptor
l~-adrenoceptor
Noradrenaline '
release
Endothelin actions
See Fig. 2
Phosphoinositide system. The PI cycle is the most recently identified yet most thoroughly characterized second messenger system. The complex elements of the PI cycle 18 can only be briefly summarized here. Stimulation of reTIN& Vol. 13, No. 6, 1990
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ceptors by neurotransmitters or hormones triggers the G protein-activated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C. The products of this reaction are inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C, which in turn phosphorylates regulatory proteins. IP3 activates the release of Ca 2+ from non-mitochondrial stores, triggering numerous CaZ+-dependent reactions. IP3 can be further phosphorylated to inositol 1,3,4,5-tetrakisphosphate (IP4), whose functions are still unclear, as are those of a number of other recently identified inositol polyphosphates. The site of action of IP3 has been clarified by studies of its receptor protein. IP3 binds saturably to membranes in peripheral tissues 19 and brain 2°. An IPzbinding protein in the cerebellum has been purified to homogeneity, antisera developed, and immunohistochemical localizations conducted at light microscopic and electron microscopic levels 2Lzz. Like other elements of the PI cycle, the IP3 receptor is highly concentrated in the cerebellum, where it is selectively localized to Purkinje cells. Within Purkinje cells, the receptor is predominantly associated with the endoplasmic reticulum (ER), including both rough and smooth ER in both cell bodies and dendrites 22,23. Recently, a cDNA for a protein called P400, which is missing in several mutant mice lacking cerebellar Purkinje cells, has been cloned from a mouse cDNA library24. The expressed recombinant protein has IP3binding activity, and thus appears to be a Purkinje cell IP3 receptor. It has a number of regions homologous to the ryanodine receptor, the Ca2+ channel of skeletal muscle sarcoplasmic reticulum 25. In keeping with the potential importance of Ca2+ in Purkinje cells, Ca2+-associated proteins are also concentrated in the cerebellum, particularly in Purkinje ceils. Thus, the Ca2+-activated ATPase, which pumps Ca 2+ into the endoplasmic reticulum, is highly enriched in the cerebellum, especially in Purkinje cells 26, as are Ca2+-binding proteins such as calbindin 28, parvalbumin, calcineurin, a Ca2+-dependent protein phosphatase, and an isoform of calmodulindependent protein kinase II (Refs 27, 28). Dendrites of Purkinje cells possess a Ca2+ action potential 7,8. We have studied the distribution in situ of intracellular Ca2+ stores by incubating brain sections with 45Ca2+ and ATP in conditions in which only ER stores can accumulate Ca2+ (Re£ 29). IP3 releases accumulated 45Ca2+ from this pool with a pharmacological specificity characteristic of IP3 receptors, and the cellular loci of this release can be monitored by autoradiography of 45Ca2+ in the brain slices. About 50% of the 45Ca2+ in the Purkinje ceil ER can be released from soma and dendrites by IP3. Sites of uptake of 45Ca2+ released by IP3 in brain correspond to sites of IP3 receptors localized by autoradiography with [3H]IP3 (Ref. 20). These sites include Purkinje cells of the cerebellum, pyramidal cells especially of CA1 in the hippocampus, and regions of cerebral cortex, caudate/putamen, thalamus and substantia nigra. The similar localization of 45Ca2+ released by IP3 and IP3-binding sites suggests that the IP3-binding protein we have purified is a physiological IP3 receptor. This conclusion has been confwmed by reconstitution experiments in which purified IP3 receptor218
binding protein is incorporated into lipid vesicles that are loaded with 45Ca2+. IP3 stimulates 45Ca2+ flux in the reconstituted protein-lipid vesicles with a potency and specificity that matches that of IPa-binding sites a°. Besides establishing that the IP3-binding protein is a physiological receptor, these studies show that it contains a Ca 2+ channel as well as the IP3 recognition site. What neurotransmitter receptors provide the endogenous stimulation for the PI system in Purkinje cells? We have recently obtained pharmacological evidence suggesting the importance of the novel subtype of the glutamate receptor described above 16, which we term the Quis-PI type (Fig. 2). Experiments using lesions and mutant mice that lack either Purkinje cells (Purkinje cell degeneration, pcd) or granule cells (weaver) strongly suggest that these receptors are postsynaptic to the parallel fibers from granule cells 31. This explains the great enrichment of components of the PI cycle in Purkinje cells, since the synapse from granule cell to Purkinje cell is quantitatively the predominant one in cerebellar cortex. Cyclic nucleotides. Endogenous cyclic GMP (cGMP), guanylate cyclase, and cGMP-dependent protein kinase are highly concentrated in Purkinje cells32-34. Glutamate augments levels of cGMP in the cerebellum via receptors that are probably different from the ionotropic and PI sites 35,36. Climbing fiber activation dramatically enhances cGMP levels in Purkinje cells 36,37 suggesting that cGMP mechanisms may be associated with climbing fibers. Cyclic AMP is also present in Purkinje cells, but its concentration is substantially less than in other parts of the brain3z. A cyclic nucleotide phosphodiesterase is enriched in Purkinje cells, and induced by the presence of intact parallel fibers 38. Cyclic AMP is presumably a second messenger for the locus coeruleus noradrenergic input to Purkinje cells, which is mediated by [3-adrenoceptors39. However, most cerebellar adenylate cyclase, as demonstrated by forskolin binding, appears to be associated with granule cell parallel fibers and terminals, and inhibited by adenosine A1 receptors (see below). Intercellular m e s s e n g e r s While intracellular second messenger molecules have been appreciated for more than three decades, the notion of intercellular messengers for neurotransmitters is more recent. Some of these, such as adenosine, appear to have relatively traditional modulatory roles. Others, such as nitric oxide, may have very non-traditional methods of synthesis, transitcation and action. Adenosine. Adenosine is often termed a 'neuromodulator' or 'putative neurotransmitter '4°. Some lines of evidence favor a conventional neurotransmitter role for adenosine. It is stored in specific neuronal populations in the brain 41, can be released upon depolarization of neurons in a Ca2+-dependent fashion 42, and exerts actions on other ceils that mimic known synaptic events 43. On the other hand, adenosine levels can vary several orders of magnitude depending on a tissue's state of oxygenation, so that in organs such as the heart it is thought of as a sensor of hypoxia which homeostatically regulates blood vessel diameter and cardiac dynamics 44. In the cerebellum, a synaptic role for adenosine as a TINS, VoL 13, No. 6, 1990
Locus coeruleus fiber (noradrenaline)
I]-adrenoceptor
Phospholipase C " ' " m"
cAMP
(v) ~:~ Protein k//inaseA (Ca subunit)
Parallel fiber terminal / / (glutamate)
~7 p IP3 Quis-PI Calmedinkk C a 2 + / P glutarrlate receptor ~"
IP3 receptor ~~~C~2T~
C~~~~----~
Ca +ATPase l\ (slow twitch/ \ ~ cardiac isoforms)
i//
///
Fig. 2. Model of the PI system in Purkinje cells. As discussed in the text, we propose that glutamate released from parallel fibers activates phospholipase C via a G protein, probably Go. PLC~ is a major form in Purkinje cells and thus may be the isozyme responsible. Protein kinase C is activated by diacylglyceroL IPz induces the IP3 receptor to release Ca2+ from intracellular stores. Calmedin is a membrane-associated protein that conveys the ability o f Ca 2+ to inhibit IP3 binding to its receptor 95. Calcium can be pumped into the El? via an isoform of the slow twitch~cardiac Ca2+-A TPase. Some elements of the ER contain Ca2+ that cannot be released by IP3; presumably these elements have no IP3 receptors. The PI response is modulated by cAMP, activated by (among other neurotransmitters) noradrenafine released from terminals of locus coeruleus fibers. The cAMP-dependent protein kinase (protein kinase A) phosphorylates the IP3 receptor, decreasing its affinity for IP3. This effect may be a molecular basis for crosstalk between the two second messenger systems. Cyclic AMP is presumably formed at noradrenergic synapses in Purkinje cell somata, while synaptic PI responses occur in dendrites so that the spatial basis for crosstalk is unclear, although high densities of IP3 receptors occur in the somata. Abbreviations: DAG, diacylglycerol; Go and G,, GTP-binding proteins; IPz, mositol 1,4,5trisphosphate; P, phosphate group; PIP2, phosphatidyfinositol 4,5-bisphosphate; Quis-PI, quisqualate PI-stimulating subtype of glutamate receptor. modulator of other neurotransmitters can be supported on the basis of specific localizations and actions. Adenosine inhibits the release of other neurotransmitters, especially excitatory neurotransmitters such as glutamate, in the cerebellum as well as other brain regions 45. Autoradiographic studies have localized adenosine A1 receptors to parallel fibers, as receptors are depleted in mutant mice that lack granule cells 46. Adenylate cyclase has also been identified on parallel fibers using forskolin binding 47. Endogenous adenosine has been localized by immunohistochemical techniques to Purkinje cells and their dendrites 41. Thus, endogenous adenosine released from Purkinje cell dendrites might selectively influence glutamate release from adjacent parallel fiber terminals via a cAMP-dependent mechanism. Adenosine might accumulate extracellularly during the relative hypoxia associated with the high firing rates of Purkinje cells, and thus inhibit further release of glutamate from parallel fibers, to diminish excessive excitation of Purkinje cells. Taurine. Taurine may have a similar transmitter or modulator function in the cerebellum. Taurine and a marker enzyme in its metabolic pathway, cysteine sulfuric acid decarboxylase, were localized by immunohistochemistry to subsets of Purkinje cells TINS, VoL 13, No. 6, 1990
located in broad parasagittal bands, as well as to cerebellar intemeurons 48. Taurine is released in a Ca2+-dependent fashion 49 and exerts a hyperpolarizing effect, perhaps by preventing dendritic influx of Ca 2+ (Ref. 50). It can also modulate the release of other neurotransmitters 51. It has been hypothesized that it is released by Purkinje cells, particularly from their dendrites, to influence other Purkinje cells or afferent presynaptic terminals 48. Endothelin. Two very recently identified intercellular messengers, which were originally characterized in peripheral blood vessels, are the peptide endothelin, an endothelium-derived contracting factor, and nitric oxide, also known as endothelium-derived relaxing factor. Endothelin is a potent vasoconstricting 21-amino acid peptide, which is released in the periphery by endothelial cells, and causes the contraction of closely adjacent smooth muscle cells in blood vessels 52. Molecular cloning studies have now established that there are three closely related endothelin peptides, termed ET1, 2 and 3 (Ref. 53). Endothelin binds with high affinity to specific receptors and potently stimulates the PI cycle54. In brain slices, endothelin also stimulates the PI system, with pronounced effects apparent in the cerebellum 54.
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In the periphery, we have identified sites of synthesis of endothelin in vascular endothelial cells, respiratory epithelial cells of bronchioles, and other cells using in situ hybridization and RNA blot analysis 55. Several cell types in the brain can synthesize endothelin. We find that ET1 mRNA can be expressed by primary glial cells from postnatal rat cerebellum56; however, in adult rat brain ET3 mRNA is expressed to a greater extent than ET1 (Ref. 55). Giaid et al. 57,58 have localized endothelin to Purkinje cells and other neurons using in situ hybridization and inununohistochemistry. Endothelin may act upon both neurons and glia. Endothelin induces PI turnover in primary cultures of cerebellar granule cells n9 but endothelin-stimulated PI turnover is not reduced in nervous, pcd or weaver mice 56. Furthermore, endothelin stimulates PI turnover in C6 glioma cells and primary cerebellar glial cells 56. Finally, microscopic visualization of cytoplasmic Ca2+ using the Ca2+-sensitive dye fura-2 reveals an endothelininduced mobilization of Ca2+ in both C6 glioma and primary cerebellar glia6°. We have recently found that endothelin is a mitogen for cultured C6 glioma cells or primary glial cultures 56, raising the possibility that endothelin, in part released from glial cells themselves, potentiates glial proliferation in vivo during development or reactive gliosis. Nitric oxide. Perhaps the most remarkable messenger molecule is the recently identified nitric oxide (NO). NO (originally called endotbelium-derived relaxing factor or EDRF) is formed by endothelial cells in blood vessels, and when released it relaxes smooth muscle cells closely adjacent in the blood vessel wall61,62. In cerebellar preparations, Garthwaite63 obtained evidence suggesting the release of NO, possibly by granule cells. NO stimulates guanylate cyclase and so may regulate the formation of cGMP in Purkinje cells of the cerebellum and/or, as suggested by Garthwaite, in gila63. We have shown that NO mediates the glutamate-induced enhancement of cGMP levels in cerebellar tissue slices 04, as observed independently by Garthwaite et al. 65. NO is derived enzymatically from arginine, with citrulline formed stoichiometrically 04,66-69. Glutamate and related amino acids enhance the conversion of arginine to NO and citrulline in close parallel with their augmentation of cGMP levels 64. Monomethylarginine, a potent inhibitor of the NO-forming enzyme, which is properly designated NO synthase (EC 1.14.23), blocks glutamate-elicited formation of cGMP. This effect is reversed by excess arginine. The properties of NO synthase indicate a major regulatory role for Ca2+. The purified enzyme is absolutely dependent on Ca2+ (ECso = 200 riM) and calmodulin (EC50 = 10 riM)69. Thus, NO synthesis would be activated by Ca2+ levels associated with IP3 actions or with the opening of cell membrane Ca2+ channels. Unlike classic neurotransmitters, NO does not appear to be stored and released in a Ca2+-dependent fashion by exocytosis. Instead, NO may diffuse locally across cell membranes to activate guanylate cyclase in adjacent cells, in analogy with its actions in endothelium and smooth muscle. NO activates soluble guanylate cyclase by binding to iron in the heme portion of the enzyme; it is highly reactive and might influence other metal-associated sites, and is rapidly converted to NOz- and NO3- (t~ is 5 s at 25°C). 220
We have recently purified NO synthase to homogeneity based on a simple, sensitive and specific assay monitoring the conversion of [3H]arginine to [3H]citrulline and on our discovery that the enzyme requires calmodulin for activity69. With antisera to NO synthase, we have localized the enzyme immunohistochemically throughout the brain and periphery (Bredt, D., Hwang, P. and Snyder, S. H., unpublished observations). In the brain, NO synthase occurs primarily in neurons and also in the endothelium of large blood vessels with no giial localizations. The neuronal localizations are discrete. Thus, in the cerebellum, NO synthase occurs in basket cells and their horizontal processes that synapse on Purkinje cells, and in mossy fibers that synapse on granule cells. We find no evidence for the enzyme occurring in granule cells, as has been suggested by Garthwaite63. Since NO synthase-containing fibers make close contact with other neurons, it seems unlikely that glia are a major target of NO actions. The synapses of basket cells upon Purkinje cells, which have high guanylate cyclase activity, can explain enhanced cGMP formation in response to NO. Other brain areas selectively enriched in NO synthase include the granule cell layer of the olfactory bulb, the superior and inferior colliculi, the islands of Callejae, the horizontal limb of the diagonal band of Broca, and the stria terminalis. In the periphery, NO synthase occurs in the endothelium of blood vessels and, most prominently, in autonomic nerve plexuses. For instance, the enzyme is concentrated in the nerve plexus of the retina, which innervates the choroid and retinal pigmented epithelium, and sympathetic fibers in the adrenal medulla. We have not detected NO synthase in macrophages, perhaps because the macrophage enzyme, whose co-factor requirements differ from the brain and endothelial enzyme, does not react with our antiserum. Messenger molecules - potential for selectivity and plasticity If one takes into consideration second messenger and intercellular messenger molecules, enzyme and receptor subtypes, as well as neurotransmitters, the resultant 'wiring diagram' of the cerebellum is considerably enriched in potential selectivity of signal communication. Both parallel and climbing fibers excite Purkinje cells via direct ionic mechanisms as well as second messenger effects. Both climbing and parallel fibers activate Purkinje cells with complex patterns of increases in the permeability of Ca2+ and sodium which may involve Ca~+-dependent dendritic spikes 7,8. Differential roles of ionotropic and second messenger-linked receptors in synaptic communication in the cerebellum are still uncertain. Electrophysiological responses in Purkinje cells to stimulation of parallel fibers are blocked by the glutamate antagonist kynurenate, and the quisqualate antagonist CNQX n, indicating a role for the quisqualate subtype of ionotropic receptor and ruling out a direct role for the Quis-PI receptor, which is unaffected by CNQX 1a,7°. The two forms of response to parallel fiber activation may have different functions, with PI turnover conveying trophic or other information. Aspartic acid, which may mediate climbing fiber TINS, Vol. 13, No. 6, 1990
effects, is antagonized by APV (aminophosphonovalerate), suggesting an NMDA-related receptorlL Given the selectivity implicit in the subtypes of ionotropic glutamate responses and the dendritic spatial complexity available for different cerebellar afferents, why is there such a plethora of second messengers as well? Second messenger systems generally exert slower, more sustained effects than ionotropic systems 71. Second messengers provide more opportunities for convergence and divergence between different receptors and ion channels 71. Further, an abundance of molecular entities, each with a specific distribution, affords a greater potential for selective regulation. Thus, the PI cycle involves a receptor coupled to a G protein, which activates phospholipase C (PLC), generating two second messengers, one mobilizing Ca2+ stores, the other activating protein kinase C 73 (Fig. 2). Messenger RNA and protein for isozymes of PLC have very different distributions in brain, with one isozyme (PLC~) highly localized to Purkinje cells in cerebellum, while others are present at lower levels in both Purkinje cells and granule cells 72-74. Isozymes of PKC also have different distributions 75. Each of these molecules may be regulated by different mechanisms. The differential distribution of GTP-binding protein G~ isoforms and their mRNAs has provided clues to their functions 76,77. Gs and Gi mediate, respectively, stimulation and inhibition of adenylate cyclaseTM. However, the identity of the G protein associated with stimulation of PI turnover has until recently been uncertain. Based on a similarity between the distributions of Go and IP3 receptors, we suggested that Go might activate the PI cycle in brain 76,77,79. Recently, Moriarty et al. 80 have provided direct evidence that Go is responsible for the PI-mediated electrophysiological response to acetylcholine in Xenopus oocytes. They found that purified Go reconstitutes the response to acetylcholine in pertussis toxin-treated oocytes, and that G,o subunits activated by GTP-¥-S directly evoked the electrophysiological response. There is a greater potential for interactions and crosstalk among different afferents where there are more molecular nodal points. A link between cAMP and PI second messenger systems in Purkinje cells is suggested by our observations that the IP3 receptor can be phosphorylated by cAMP-dependent protein kinase 81 (see Fig. 2). This phosphorylation does not markedly alter the affinity of IP3 for its binding sites; however, it renders IP3 only one-tenth as potent in releasing Ca2+. Additionally, cAMP-dependent phosphorylation of the Ca2+ pump in Purkinje cells enhances the uptake of 45Ca2+ into Purkinje cells in brain sections, so that IP3 acts upon a pool of 45Ca2+ about three times larger than in the unphosphorylated state. Thus, the interaction between cAMP and the IP3 receptor under physiological circumstances may be complex. Finally, interactions among the messenger molecules described above may have a role in synaptic plasticity. In the cerebellum, long-term depression (LTD) is in some respects analogous to long-term potentiation (LTP) in the hippocampus. In the CA1 region of the hippocampus the conjunction of two specific synaptic inputs to a pyramidal cell strengthens one of them, so that its subsequent input to that pyramidal cell is potentiated, leading to greater TINS, VoL 13, No. 6, 1990
excitation of the neuron to which the pyramidal cell projects. In the cerebellum, the conjunction of parallel fiber and climbing fiber stimulation leads to depression of the ability of the parallel fiber to activate the Purkinje cells2. Since the output of the Purkinje cells is inhibitory, the net effect, as in the hippocampus, will be increased firing of the Purkinje cell's target neuron. The receptor at the parallel fiber synapse in LTD is of the quisqualate ionotropic or metabotropic type 83, unlike hippocampal LTP in CA1 and dentate gyrus, which involves the NMDA receptor. One might predict that LTD would involve decreased ability of the parallel fiber to stimulate PI turnover. In the hippocampus, there would appear to be two phases to LTP, the first presynapfic, the second postsynaptic 84. This has led to the hypothesis that trophic factors or neuromodulators transfer information during the course of LTP, first from the postsynaptic cell to the presynaptic terminal, then from the presynaptic terminal back again s5. Williams and Bfiss86 have suggested that arachidonic acid acts as a retrograde messenger from the postsynaptic induction site to the presynaptic terminal in hippocampus; perhaps it also has a role in LTD in the cerebellum. Other modulators, such as NO, adenosine or taurine, could also be involved. Long-term plastic changes in neurons probably involve alterations in transcription of specific genes, as have been described for growth factors, acting via various second messenger systems 87-s9. Related actions are mediated by cellular oncogenes, several of which are present in high concentrations in brain 9°,91. Several oncogenes are selectively expressed in particular cells in cerebellum: H-ras protein is localized in Purkinje cells of adult rats, as well as in the cerebral cortex 92. Purkinje cells of adult chicken contain more pp62c-yes than any other cells in brain 9°. Cerebellar transcripts of c-myc can similarly be localized almost entirely to Purkinje cells 93. Neurotransmitter receptors, such as the serotonin 1C receptor, can, under certain circumstances, be oncogenes 94. As noted above, endothelin can be mitogenic for glial cells. The previously sharp boundaries between neurotransmitters, neuromodulators and trophic factors are thus becoming increasingly blurred. The cerebellum, with its defined synaptic connections, should remain an excellent model to help unravel these complex molecular interactions.
Selected references 1 Llin~.s, R. R. (1975) Sci. Am. 232, 56-71 2 Ito, M. (1984) The Cerebellum and Neural Control Raven Press 3 Wuenschell, C. W., Fisher, R. S, Kaufrnan, D. L. and Tobin, A. J. (1986) Proc. NatlAcad. Sci. USA 83, 6193-6197 4 Sieghart, W. (1989) Trends Pharmacol. Sci. 10, 407-411 5 Bowery, N. (1989) Trends Pharmacol. Sci. 10, 401-407 6 Pritchett, D. B. et al. (1989) Nature 338, 582-585 7 Llin~.s, R. and Sugimori, M. (1980) J. Physiol. (London) 305, 197-213 8 Tank, D. W., Sugimori, M., Connor, J. A. and Llin~s, R. R. (1988) Science 242,773-777 9 Sornogyi, P., Halasy, K., Somogyi, J., Storrn-Mathisen, J. and Ottersen, O. P. (1986) Neuroscience 19, 1045-1050 10 Olson, J. M. M., Greenamyre, J. T., Penney, J. B. and Young, A. B. (1987) Neuroscience 22, 913-923 11 Hirano, T. and Hagiwara, S. (1988) Proc. NatlAcad. Sci. USA 85, 934-938 12 Wiklund, T., Toggenburger, G. and Cuenod, M. (1982) Science 216, 78-80 221
Acknowledgements Supported by a grant of the International Life SciencesInstitute, Public Health Service Grants MH- 18501, DA-00266, Research ScientistAward DA00074 to SHS, MH43040 to CAR, Training Grant GM07309 to DSB and a
gift from BristolMyers Squibb Company. CAR is a Pew Scholar in the Biomedical Sciences.
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13 Grandes, P., Cuenod, M. and Streit, P. (1989) Soc. Neurosci. Abstr. 15, 941 14 Monaghan, D. T., Bridges, R. J. and Cotman, C. W. (1989) Annu. Rev. Pharmacol. Toxicol. 29, 365-402 15 Sladeczek, F., R~casens, M. and Bockaert, J. (1988) Trends Neurosci. 11,545-549 16 Sigiyama, H., Ito, I. and Hirono, C. (1987) Nature 325, 531-533 17 Murphy, S. N. and Miller, R. J. (1988) Proc. NatlAcad. Sci. USA 85, 8737-8741 18 Berridge, M. J. and Irvine, R. F. (1989) Nature 341,197-205 19 Spat, A., Bradford, P. G., McKinney, J. S., Rubin, R. P. and Putney, J. W., Jr (1986) Nature 319, 514-516 20 Worley, P. F., Baraban, J. M., Colvin, J. S. and Snyder, S. H. (1987) Nature 325, 159-161 21 Suppattapone, S., Worley, P. F., Baraban, J. M. and Snyder, S. H. (1988)J. Biol. Chem. 263, 1530-1534 22 Ross, C. A. etal. (1989) Nature 339, 468-470 23 Mignery, G. A., Sudhof, T. C., Takei, K. and De Camilli, P. (1989) Nature 342, 192-195 24 Furuichi, T. etal. (1989) Nature 342, 32-38 25 Takeshima, M. et al. (1989) Nature 339, 439-445 26 Kaprielian, Z., Campbell, A. M. and Fambrough, D. M. (1989) Mol. Brain Res. 6, 55-60 27 Walaas, S. I. etal. (1988) Mol. Brain Res. 4, 233-242 28 Jande, S. S., Maler, L. and Lawson, D. E. M. (1981) Nature 294, 765-767 29 Verma, A., Ross, C. A., Supattapone, S., Verma, D. and Snyder, S. H. (1989) Soc. Neurosci. Abstr. 15, 658 30 Ferris, C. D., Huganir, R. L., Supattapone, S. and Snyder, S. H. (1989) Nature 342, 87-89 31 Blackstone, C. D., Supattapone, S. and Snyder, S. H. (1989) Proc. Natl Acad. Sci. USA 86, 4316-4320 32 Ferrendelli, J. A. (1978) Adv. Cyclic Nucleotide Res. 9, 453-464 33 Lohmann, S. M., Walter, U., Miller, P. E., Greengard, P. and De Camilli, P. (1981) Proc. NatlAcad. Sci. USA 78, 653-657 34 Nakane, M., Ichikawa, M. and Deguchi, T. (1983) Brain Res. 273, 9-15 35 Novetli, A. and Henneberry, R. C. (1987) Dev. Brain Res. 34, 307-310 36 Nicoletti, F. etal. (1986)J. Neurosci. 6, 1905-1921 37 Biggio, G., Brodie, B. B., Costa, E. and Guidotti, A. (1977) Proc. Natl Acad. Sci. USA 74, 3592-3596 38 Balaban, C. D., Billingsley, M. L. and Kincaid, R. L. (1989) J. Neurosci. 9, 2374-2381 39 Siggins, G. R., Oliver, A. P., Hoffer, B. J. and Bloom, F. E. (1971) Science 171, 192-194 40 Snyder, S. H. (1985) Annu. Rev. Neurosci. 8, 103-124 41 Braas, K. M., Newby, A. C., Wilson, V. S. and Snyder, S. H. (1986) J. Neurosci. 6, 1952-1961 42 Pull, I. and Mcllwain, H. (1977) Neurochem. Res. 2,203-216 43 Van Calker, D., Muller, M. and Hamprecht, B. (1979) J. Neurochem. 33, 999-1005 44 Berne, R. M. (1980) Circ. Res. 46, 807-813 45 Fredholm, B. B. and Dunwiddie, T. V. (1989) Trends PharmacoL Sci. 9, 130-134 46 Goodman, R. R., Kuhar, M. J., Hester, L. and Snyder, S. H. (1983) Science 220, 967-969 47 Worley, P. F., Baraban, J. M., De Souza, E. B. and Snyder, S. H. (1986) Proc. NatlAcad. Sci. USA 83, 4053-4057 48 Magnusson, K. R. eta/. (1988) J. Neurosci. 8, 4551-4564 49 Lehmann, A., Lazarewicz, J. W. and Zeise, M. (1985) J. Neurochem. 45, 1172-1177 50 Okamoto, K., Kimura, H. and Sakai, Y. (1983) Brain Res. 260, 261-269 51 Kuriyama, K. (1980) Fed. Proc. 39, 2680-2684 52 Yanagisawa, M. etaL (1988) Nature 332,411--415 53 Inoue, A. et aL (1989) Proc. Natl Acad. Sci. USA 86, 2863-2867 54 Ambar, I., Kloog, Y., Schwartz, I., Hazum, E. and Sokolovsky, M. (1989) Biochem. Biophys. Res. Commun. 158, 195-201 55 MacCumber, M. W., Ross, C. A., Glaser, B. M. and Snyder, S. H. (1989) Proc. Natl Acad. 5ci. USA 86, 7285-7289 56 MacCumber, M. W., Ross, C. A. and Snyder, S. H. Proc. Natl Acad. Sci. USA (in press) 57 Giaid, A. et al. (1989) Proc. Natl Acad. 5ci. USA 86, 7634-7638 58 Giaid, A. et al. (1989) Soc. Neurosci. Abstr. 15, 187 59 Lin, W. W., Lee, C. Y. and Chuang, D-M. (1989) Eur. J. Pharmacol. 166, 581-582
60 Supattapone, S., Simpson, A. W. M. and Ashley, C. C. (1989) Biochem. Biophys. Res. Commun. 165, 1115-1122 61 Moncada, S., Radomski, M. W. and Palmer, R. M. J. (1988) Biochem. Pharmacol. 37, 2495-2501 62 Furchgott, R. F. and Vanhoutte, P. M. (1989) FASEB J. 3, 2007-2018 63 Garthwaite, J., Charles, S. J. and Chess-Williams, R. (1988) Nature 336, 385-388 64 Bredt, D. S. and Snyder, S. H. (1989) Proc. Natl Acad. Sci. USA 86, 9030-9033 65 Garthwaite, J., Garthwaite, G., Palmer, R. M. J. and Moncada, S. (1989) Eur. J. PharmacoL 172,413-416 66 Hibbs, J. B., Jr, Taintor, R. R. and Vavrin, Z. (1987) Science 235, 473-476 67 Marietta, M. A., Yoon, P. S., lyengar, R., Leaf, C. D. and Wishnock, J. S. (1988) Biochemistry 27, 8706-8711 68 Knowles, R. G., Palacios, M., Palmer, R. M. J. and Moncada, S. (1989) Proc. Natl Acad. Sci. USA 86, 5159-5162 69 Bredt, D. S. and Snyder, S. H. (1990) Proc. Natl Acad. Sci. USA 87, 682-685 70 HonorS, T. et aL (1988) Science 241,701-703 71 Nicoll, R. A. (1988) Science 241,545-551 72 Rhee, S. G., Suh, P-G., Ryu, S-H. and Lee, S. Y. (1989) Science 244, 546-550 73 Nishizuka, Y. (1988) Nature 334, 661-665 74 Ross, C. A., MacCumber, M. W., Glatt, C. E. and Snyder, S. H. (1989) Proc. NatlAcad. Sci. USA 86, 2923-2927 75 Huang, F. L., Yoshida, Y., Nakabayashi, H., Young, S. W., III and Huang, K-P. (1988) J. Neurosci. 8, 4734-4744 76 Worley, P. F., Baraban, J. M., Van Dop, C., Neer, E. J. and Snyder, S. H. (1986) Proc. Natl Acad. Sci. USA 83, 4561--4565 77 Largent, B. L., Jones, D. T., Reed, R. R., Pearson, R. C. A. and Snyder, S. H. (1988) Proc. Natl Acad. Sci. USA 85, 2864-2868 78 Gilman, A. G. (1989) J. Am. Med. Assoc. 262, 1819-1825 79 Worley, P. F., Baraban, J. M. and Snyder, S. H. (1989) J. Neurosci. 9, 339-346 80 Moriarty, T. M., Padrell, E., Ornri, G., lyengar, R. and Landau, E. M. (1990) Nature 343, 79-82 81 Supattapone, S. eta/. (1988) Proc. NatlAcad. ScL USA 85, 8747-8750 82 Ito, M. (1989) Annu. Rev. Neurosci. 12, 85-102 83 Kano, M. and Kato, M. (1987) Nature 325, 276-279 84 Davies, S. N., Lester, R. A. J., Reymann, K. G. and Collingridge, G. L. (1989) Nature 338, 500-503 85 Stevens, C. F. (1989) Nature 338, 460-461 86 Williams, J. H. and Bliss, T. V. P. (1988) Neurosci. Left. 88, 81-85 87 Cole, A. J., Saffen, D. W., Baraban, J. M. and Worley, P. F. (1989) Nature 340, 474-476 88 Goelet, P., Castetlucci, V. F., Schacher, S. and Kandel, E. R. (1986) Nature 322, 419-422 89 Berridge, M. (1986) Nature 323,294-295 90 Sudol, M. (1988) Brain Res. Rev. 13,391-403 91 Ross, C. A., Wright, G. E., Resh, M. D., Pearson, R. C. A. and Snyder, S. H. (1988) Proc. Natl Acad. Sci. USA 85, 9831-9835 92 Tanaka, T. etal. (1987) Mol. Cell. Biochem. 75, 23-32 93 Ruppert, C., Goldovitz, D. and Wille, W. (1986) EMBOJ. 5, 1897-1901 94 Julius, D., Livelli, T. J., Jessell, T. M. and Axel, R. (1989) Science 244, 1057-1062 95 Danoff, S. K., Supattapone, S. and Snyder, S. H. (1988) Biochem. J. 254, 701-705
Letters to the Editor Letters to the editor of Trends in Neurosciences concerning recently published articles in the journal are welcomed. Please mark clearly whether they are intended for publication. Please address letters to: Editor, Trends in Neurosciences, Elsevier Publications, 68 Hills Road, Cambridge CB2 1 LA, UK.
TINS, VoL 13, No. 6, 1990
ChristopherA. Ross, DavidBredtand SolomonH. Snyder are at the Departmentsof Neuroscience, Pharmacologyand MolecularSciences, Psychiatryand BehavioralSciences, TheJohnsHopkins UniversitySchoolof Medicine, 725 North Wolfe Street, Baltimore,MD 21205, USA.
As data accumulate, the mammalian brain reveals its complex and subtle synaptic mechanisms. In the simplest system, a neurotransmitter binds to the receptor portion of a molecular complex incorporating an ion channel and thus alters the membrane potential, leading to excitatory or inhibitory effects. In more complex systems, receptors are coupled to second messenger systems to generate signals of longer duration and to modulate more diverse molecular mechanisms. The cerebellar cortex has a relatively simple wiring diagram with the primary neurotransmitter of most inhibitory and excitatory synapses well established. The second messenger signalling systems are more complex and those of the cerebellar output, the Purkinje cells, are the best characterized. More recently, molecules that might act as neuromodulators, carrying messages between neurons and between neurons and glial cells, have been identified, such as endothelin and nitric oxide. The classic neurotransmitters and novel neuromodulators, together with second messenger-activated trophic factors, can interact in complex ways; in this review Christopher Ross, David Bredt and Solomon Snyder discuss how studies of cerebellar circuitry and biochemistry are revealing such interrelations. The synaptic diversity that permits complex information transfer in the brain has often been ascribed to its hundred or more putative neurotransmitters. Because only a handful of second messenger systems exist, they have not been a major focus for the exploration of synaptic subtleties. However, recent demonstrations of marked molecular heterogeneity and regional specificities within the cAMP and phosphoinositide (PI) systems provide rich possibilities for signal discrimination. Novel 'intercellular' messengers, such as nitric oxide and adenosine, afford unprecedented ways for regulating neuronal communication. The cerebellum is an ideal site for investigating these 'messenger molecules'. Thus, elements of the PI cycle, such as the inositol 1,4, 5-trisphosphate (IPz) receptor, are enriched several hundred-fold in the cerebellum compared with other brain regions or peripheral tissues. Moreover, neuronal connectivity in the cerebellum is surprisingly straightforward. Accordingly, we focus on the cerebellum to review recent advances in biochemical messenger systems in brain function.
Cerebellar wiring diagram and neurotransmitters The relative simplicity of the wiring diagram of the cerebellar cortex has made it a favored site for analysing specific synaptic interactions 1,2. The cerebellar cortex has only three major neuronal inputs and a single neuronal output (Fig. 1). One input is via mossy fibers, with cell bodies located in the spinal cord, the pontine nuclei, and other sites, and terCorrespondence should be addressedto Solomon H. Snyder. 216
© 1990, ElsevierSciencePublishersLtd,(UK)
minals on specialized granule cell dendritic complexes called cerebellar glomeruli. Granule cells then contact dendrites of the Purkinje cells via excitatory parallel fibers, which release glutamate. The second input is via climbing fibers, whose cell bodies are mostly in the inferior olive of the brain stem and whose axons terminate directly on individual Purkinje cells. A third, modulatory input arises from monoaminergic neurons in the locus coeruleus and raphe nuclei, with terminals predominantly on Purkinje cells. The output of the cerebellum derives from Purkinje cells, whose axons project to the brain stem and deep cerebellar nuclei. The two main cerebellar inputs, climbing fibers and mossy fibers, are excitatory, while Purkinje cells, which contain GABA, are inhibitory. There are three major interneurons, all capable of synthesizing GABA3 and thus inhibitory, in the cerebellar cortex - basket cells, stellate cells and Golgi type II cells. Basket and stellate cells, whose perikarya lie in the molecular layer, receive afferents from parallel fibers and inhibit Purkinje cells. Golgi cells, whose perikarya are in the granule cell layer, receive impulses from parallel fibers, as well as direct connections from mossy fibers. Their output, also inhibitory, feeds back to the cerebellar glomeruli, and thus influences the transfer of information from the mossy fibers to granule cells. Presumably, GABA released from stellate, basket and Golgi II cells directly opens chloride ion channels via GABA receptors. Autoradiographic studies show GABAA receptors to be most concentrated in the granule cell layer, and GABAB receptors in the molecular layer 4,5. In addition, the 72 subunit of the GABAA receptor, important for benzodiazepine binding, is expressed by Purkinje cells6. As the sole neuronal output of the cerebellum, the Purkinje cell is the final common path of cerebellar activity. It is also uniquely enriched in elements of the PI cycle, which may relate to certain of its properties. For instance, a single Purkinje cell receives as many as 200 000 synaptic junctions, the most of any central neuron. These inputs come predominantly from parallel fibers of granule cells, which employ a PIlinked glutamate receptor (see below). Additionally, Purkinje cell dendrites display prominent action potentials dependent on calcium (Ca2+). Granule cells comprise some 90% of all cerebellar neurons and are the only intrinsic neurons with an excitatory transmitter 1,2. Their parallel fibers synapse exclusively upon the dendritic spines of Purkinje cells, each one innervating the distal dendrites of many Purkinje cells. Because of the great abundance of granule cells, the parallel fiber-Purkinje cell synapse is quantitatively the predominant synapse in the cerebeilar cortex. By contrast, each climbing fiber invests the soma and proximal dendrites of a single Purkinje cell, like a vine enveloping a tree; climbing fibers powerfully excite Purkinje cells, with Ca 2+dependent plateau potentials and multiple spikes 7,s. The actions of excitatory transmitters in the cerebellum appear to be complex. Multiple lines of
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Fig. 1. Neurotransmission in the cerebellar cortex. (A) Simpfified wiring diagram and neurotransmitters. Basket and stellate cells are distinct but, for simplicity's sake, are indicated as interchangeable as they have similar inputs and outputs and use the same transmitter. (B) 5elected receptors and second messenger systems discussed in the text. For visual simpficity, the basket cell is not shown in (B). It synthesizes NO, which presumably diffuses to the Purkinje cell to activate cGA4P formation. In (A) and (B) dark red and light red structures are inhibitory. Black and grey structures are excitatory (except the glial cell, which is indeterminate). Abbreviations: GABA, 7-aminobutyric acid; 5-HT, serotonin; NE, noradrenaflne; Quis-PI, quisqualate PI-stimulating subtype of glutamate receptor.
Parallel fiber (glutamate)
A
Basket or stellate cell (GABA)
Purkinje cell (GABA)
Granule cell (glutamate)
- " •Glial cell
Climbing fiber (excitatory amino acid) input from • medulla J
evidence indicate that glutamate Purkinje cell axon (GABA) is the transmitter of parallel output to deep fibers9-11. Fewer data are available cerebellar nuclei for climbing fibers, whose transmitter is generally thought to be aspartate or glutamate 12, though homocysteate has also been proposed 1:~. Thus far, synaptic responses at aspartate receptors have not been clearly differentiated from B Adenosine glutamate receptors. The primary receptor subtypes of glutamate receptors in the brain directly influence ion channels and are classified according Adenosine formation to the glutamate derivative that is most selective H. The subtypes are designated N-methyl-D-aspartate (NMDA; the most effective), kainate, and quisqualate. Recently, glutamate receptors that act through the PI second messenger system to mobilize intracellular cGMP Ca~+ have been identified15-17. The formation glutamate receptor subtype that stimulates PI turnover differs in pharmacological specificity from any Excitatory of the known ionotropic receptors, amino acid being most sensitive to quisrelease qualate, but insensitive to the ionotropic agonist AMPA (o~-amino-3hydroxy-5-methyl-isoazole-4-proprionate), and the antagonist CNQX (6-cyano-2, 3-dihydroxy-7nitroqukloxaline) 15-17. Intraeellular mediators
Golgi cell (GABA)
Monoaminergic fiber (NE or 5-HT) input from locus coeruleus or raphe Mossy fiber input from pons and / spinal cord
Glutamate release
See Fig. 2
~
QUIS-PI receptor
l~-adrenoceptor
Noradrenaline '
release
Endothelin actions
See Fig. 2
Phosphoinositide system. The PI cycle is the most recently identified yet most thoroughly characterized second messenger system. The complex elements of the PI cycle 18 can only be briefly summarized here. Stimulation of reTIN& Vol. 13, No. 6, 1990
217
ceptors by neurotransmitters or hormones triggers the G protein-activated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C. The products of this reaction are inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C, which in turn phosphorylates regulatory proteins. IP3 activates the release of Ca 2+ from non-mitochondrial stores, triggering numerous CaZ+-dependent reactions. IP3 can be further phosphorylated to inositol 1,3,4,5-tetrakisphosphate (IP4), whose functions are still unclear, as are those of a number of other recently identified inositol polyphosphates. The site of action of IP3 has been clarified by studies of its receptor protein. IP3 binds saturably to membranes in peripheral tissues 19 and brain 2°. An IPzbinding protein in the cerebellum has been purified to homogeneity, antisera developed, and immunohistochemical localizations conducted at light microscopic and electron microscopic levels 2Lzz. Like other elements of the PI cycle, the IP3 receptor is highly concentrated in the cerebellum, where it is selectively localized to Purkinje cells. Within Purkinje cells, the receptor is predominantly associated with the endoplasmic reticulum (ER), including both rough and smooth ER in both cell bodies and dendrites 22,23. Recently, a cDNA for a protein called P400, which is missing in several mutant mice lacking cerebellar Purkinje cells, has been cloned from a mouse cDNA library24. The expressed recombinant protein has IP3binding activity, and thus appears to be a Purkinje cell IP3 receptor. It has a number of regions homologous to the ryanodine receptor, the Ca2+ channel of skeletal muscle sarcoplasmic reticulum 25. In keeping with the potential importance of Ca2+ in Purkinje cells, Ca2+-associated proteins are also concentrated in the cerebellum, particularly in Purkinje ceils. Thus, the Ca2+-activated ATPase, which pumps Ca 2+ into the endoplasmic reticulum, is highly enriched in the cerebellum, especially in Purkinje cells 26, as are Ca2+-binding proteins such as calbindin 28, parvalbumin, calcineurin, a Ca2+-dependent protein phosphatase, and an isoform of calmodulindependent protein kinase II (Refs 27, 28). Dendrites of Purkinje cells possess a Ca2+ action potential 7,8. We have studied the distribution in situ of intracellular Ca2+ stores by incubating brain sections with 45Ca2+ and ATP in conditions in which only ER stores can accumulate Ca2+ (Re£ 29). IP3 releases accumulated 45Ca2+ from this pool with a pharmacological specificity characteristic of IP3 receptors, and the cellular loci of this release can be monitored by autoradiography of 45Ca2+ in the brain slices. About 50% of the 45Ca2+ in the Purkinje ceil ER can be released from soma and dendrites by IP3. Sites of uptake of 45Ca2+ released by IP3 in brain correspond to sites of IP3 receptors localized by autoradiography with [3H]IP3 (Ref. 20). These sites include Purkinje cells of the cerebellum, pyramidal cells especially of CA1 in the hippocampus, and regions of cerebral cortex, caudate/putamen, thalamus and substantia nigra. The similar localization of 45Ca2+ released by IP3 and IP3-binding sites suggests that the IP3-binding protein we have purified is a physiological IP3 receptor. This conclusion has been confwmed by reconstitution experiments in which purified IP3 receptor218
binding protein is incorporated into lipid vesicles that are loaded with 45Ca2+. IP3 stimulates 45Ca2+ flux in the reconstituted protein-lipid vesicles with a potency and specificity that matches that of IPa-binding sites a°. Besides establishing that the IP3-binding protein is a physiological receptor, these studies show that it contains a Ca 2+ channel as well as the IP3 recognition site. What neurotransmitter receptors provide the endogenous stimulation for the PI system in Purkinje cells? We have recently obtained pharmacological evidence suggesting the importance of the novel subtype of the glutamate receptor described above 16, which we term the Quis-PI type (Fig. 2). Experiments using lesions and mutant mice that lack either Purkinje cells (Purkinje cell degeneration, pcd) or granule cells (weaver) strongly suggest that these receptors are postsynaptic to the parallel fibers from granule cells 31. This explains the great enrichment of components of the PI cycle in Purkinje cells, since the synapse from granule cell to Purkinje cell is quantitatively the predominant one in cerebellar cortex. Cyclic nucleotides. Endogenous cyclic GMP (cGMP), guanylate cyclase, and cGMP-dependent protein kinase are highly concentrated in Purkinje cells32-34. Glutamate augments levels of cGMP in the cerebellum via receptors that are probably different from the ionotropic and PI sites 35,36. Climbing fiber activation dramatically enhances cGMP levels in Purkinje cells 36,37 suggesting that cGMP mechanisms may be associated with climbing fibers. Cyclic AMP is also present in Purkinje cells, but its concentration is substantially less than in other parts of the brain3z. A cyclic nucleotide phosphodiesterase is enriched in Purkinje cells, and induced by the presence of intact parallel fibers 38. Cyclic AMP is presumably a second messenger for the locus coeruleus noradrenergic input to Purkinje cells, which is mediated by [3-adrenoceptors39. However, most cerebellar adenylate cyclase, as demonstrated by forskolin binding, appears to be associated with granule cell parallel fibers and terminals, and inhibited by adenosine A1 receptors (see below). Intercellular m e s s e n g e r s While intracellular second messenger molecules have been appreciated for more than three decades, the notion of intercellular messengers for neurotransmitters is more recent. Some of these, such as adenosine, appear to have relatively traditional modulatory roles. Others, such as nitric oxide, may have very non-traditional methods of synthesis, transitcation and action. Adenosine. Adenosine is often termed a 'neuromodulator' or 'putative neurotransmitter '4°. Some lines of evidence favor a conventional neurotransmitter role for adenosine. It is stored in specific neuronal populations in the brain 41, can be released upon depolarization of neurons in a Ca2+-dependent fashion 42, and exerts actions on other ceils that mimic known synaptic events 43. On the other hand, adenosine levels can vary several orders of magnitude depending on a tissue's state of oxygenation, so that in organs such as the heart it is thought of as a sensor of hypoxia which homeostatically regulates blood vessel diameter and cardiac dynamics 44. In the cerebellum, a synaptic role for adenosine as a TINS, VoL 13, No. 6, 1990
Locus coeruleus fiber (noradrenaline)
I]-adrenoceptor
Phospholipase C " ' " m"
cAMP
(v) ~:~ Protein k//inaseA (Ca subunit)
Parallel fiber terminal / / (glutamate)
~7 p IP3 Quis-PI Calmedinkk C a 2 + / P glutarrlate receptor ~"
IP3 receptor ~~~C~2T~
C~~~~----~
Ca +ATPase l\ (slow twitch/ \ ~ cardiac isoforms)
i//
///
Fig. 2. Model of the PI system in Purkinje cells. As discussed in the text, we propose that glutamate released from parallel fibers activates phospholipase C via a G protein, probably Go. PLC~ is a major form in Purkinje cells and thus may be the isozyme responsible. Protein kinase C is activated by diacylglyceroL IPz induces the IP3 receptor to release Ca2+ from intracellular stores. Calmedin is a membrane-associated protein that conveys the ability o f Ca 2+ to inhibit IP3 binding to its receptor 95. Calcium can be pumped into the El? via an isoform of the slow twitch~cardiac Ca2+-A TPase. Some elements of the ER contain Ca2+ that cannot be released by IP3; presumably these elements have no IP3 receptors. The PI response is modulated by cAMP, activated by (among other neurotransmitters) noradrenafine released from terminals of locus coeruleus fibers. The cAMP-dependent protein kinase (protein kinase A) phosphorylates the IP3 receptor, decreasing its affinity for IP3. This effect may be a molecular basis for crosstalk between the two second messenger systems. Cyclic AMP is presumably formed at noradrenergic synapses in Purkinje cell somata, while synaptic PI responses occur in dendrites so that the spatial basis for crosstalk is unclear, although high densities of IP3 receptors occur in the somata. Abbreviations: DAG, diacylglycerol; Go and G,, GTP-binding proteins; IPz, mositol 1,4,5trisphosphate; P, phosphate group; PIP2, phosphatidyfinositol 4,5-bisphosphate; Quis-PI, quisqualate PI-stimulating subtype of glutamate receptor. modulator of other neurotransmitters can be supported on the basis of specific localizations and actions. Adenosine inhibits the release of other neurotransmitters, especially excitatory neurotransmitters such as glutamate, in the cerebellum as well as other brain regions 45. Autoradiographic studies have localized adenosine A1 receptors to parallel fibers, as receptors are depleted in mutant mice that lack granule cells 46. Adenylate cyclase has also been identified on parallel fibers using forskolin binding 47. Endogenous adenosine has been localized by immunohistochemical techniques to Purkinje cells and their dendrites 41. Thus, endogenous adenosine released from Purkinje cell dendrites might selectively influence glutamate release from adjacent parallel fiber terminals via a cAMP-dependent mechanism. Adenosine might accumulate extracellularly during the relative hypoxia associated with the high firing rates of Purkinje cells, and thus inhibit further release of glutamate from parallel fibers, to diminish excessive excitation of Purkinje cells. Taurine. Taurine may have a similar transmitter or modulator function in the cerebellum. Taurine and a marker enzyme in its metabolic pathway, cysteine sulfuric acid decarboxylase, were localized by immunohistochemistry to subsets of Purkinje cells TINS, VoL 13, No. 6, 1990
located in broad parasagittal bands, as well as to cerebellar intemeurons 48. Taurine is released in a Ca2+-dependent fashion 49 and exerts a hyperpolarizing effect, perhaps by preventing dendritic influx of Ca 2+ (Ref. 50). It can also modulate the release of other neurotransmitters 51. It has been hypothesized that it is released by Purkinje cells, particularly from their dendrites, to influence other Purkinje cells or afferent presynaptic terminals 48. Endothelin. Two very recently identified intercellular messengers, which were originally characterized in peripheral blood vessels, are the peptide endothelin, an endothelium-derived contracting factor, and nitric oxide, also known as endothelium-derived relaxing factor. Endothelin is a potent vasoconstricting 21-amino acid peptide, which is released in the periphery by endothelial cells, and causes the contraction of closely adjacent smooth muscle cells in blood vessels 52. Molecular cloning studies have now established that there are three closely related endothelin peptides, termed ET1, 2 and 3 (Ref. 53). Endothelin binds with high affinity to specific receptors and potently stimulates the PI cycle54. In brain slices, endothelin also stimulates the PI system, with pronounced effects apparent in the cerebellum 54.
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In the periphery, we have identified sites of synthesis of endothelin in vascular endothelial cells, respiratory epithelial cells of bronchioles, and other cells using in situ hybridization and RNA blot analysis 55. Several cell types in the brain can synthesize endothelin. We find that ET1 mRNA can be expressed by primary glial cells from postnatal rat cerebellum56; however, in adult rat brain ET3 mRNA is expressed to a greater extent than ET1 (Ref. 55). Giaid et al. 57,58 have localized endothelin to Purkinje cells and other neurons using in situ hybridization and inununohistochemistry. Endothelin may act upon both neurons and glia. Endothelin induces PI turnover in primary cultures of cerebellar granule cells n9 but endothelin-stimulated PI turnover is not reduced in nervous, pcd or weaver mice 56. Furthermore, endothelin stimulates PI turnover in C6 glioma cells and primary cerebellar glial cells 56. Finally, microscopic visualization of cytoplasmic Ca2+ using the Ca2+-sensitive dye fura-2 reveals an endothelininduced mobilization of Ca2+ in both C6 glioma and primary cerebellar glia6°. We have recently found that endothelin is a mitogen for cultured C6 glioma cells or primary glial cultures 56, raising the possibility that endothelin, in part released from glial cells themselves, potentiates glial proliferation in vivo during development or reactive gliosis. Nitric oxide. Perhaps the most remarkable messenger molecule is the recently identified nitric oxide (NO). NO (originally called endotbelium-derived relaxing factor or EDRF) is formed by endothelial cells in blood vessels, and when released it relaxes smooth muscle cells closely adjacent in the blood vessel wall61,62. In cerebellar preparations, Garthwaite63 obtained evidence suggesting the release of NO, possibly by granule cells. NO stimulates guanylate cyclase and so may regulate the formation of cGMP in Purkinje cells of the cerebellum and/or, as suggested by Garthwaite, in gila63. We have shown that NO mediates the glutamate-induced enhancement of cGMP levels in cerebellar tissue slices 04, as observed independently by Garthwaite et al. 65. NO is derived enzymatically from arginine, with citrulline formed stoichiometrically 04,66-69. Glutamate and related amino acids enhance the conversion of arginine to NO and citrulline in close parallel with their augmentation of cGMP levels 64. Monomethylarginine, a potent inhibitor of the NO-forming enzyme, which is properly designated NO synthase (EC 1.14.23), blocks glutamate-elicited formation of cGMP. This effect is reversed by excess arginine. The properties of NO synthase indicate a major regulatory role for Ca2+. The purified enzyme is absolutely dependent on Ca2+ (ECso = 200 riM) and calmodulin (EC50 = 10 riM)69. Thus, NO synthesis would be activated by Ca2+ levels associated with IP3 actions or with the opening of cell membrane Ca2+ channels. Unlike classic neurotransmitters, NO does not appear to be stored and released in a Ca2+-dependent fashion by exocytosis. Instead, NO may diffuse locally across cell membranes to activate guanylate cyclase in adjacent cells, in analogy with its actions in endothelium and smooth muscle. NO activates soluble guanylate cyclase by binding to iron in the heme portion of the enzyme; it is highly reactive and might influence other metal-associated sites, and is rapidly converted to NOz- and NO3- (t~ is 5 s at 25°C). 220
We have recently purified NO synthase to homogeneity based on a simple, sensitive and specific assay monitoring the conversion of [3H]arginine to [3H]citrulline and on our discovery that the enzyme requires calmodulin for activity69. With antisera to NO synthase, we have localized the enzyme immunohistochemically throughout the brain and periphery (Bredt, D., Hwang, P. and Snyder, S. H., unpublished observations). In the brain, NO synthase occurs primarily in neurons and also in the endothelium of large blood vessels with no giial localizations. The neuronal localizations are discrete. Thus, in the cerebellum, NO synthase occurs in basket cells and their horizontal processes that synapse on Purkinje cells, and in mossy fibers that synapse on granule cells. We find no evidence for the enzyme occurring in granule cells, as has been suggested by Garthwaite63. Since NO synthase-containing fibers make close contact with other neurons, it seems unlikely that glia are a major target of NO actions. The synapses of basket cells upon Purkinje cells, which have high guanylate cyclase activity, can explain enhanced cGMP formation in response to NO. Other brain areas selectively enriched in NO synthase include the granule cell layer of the olfactory bulb, the superior and inferior colliculi, the islands of Callejae, the horizontal limb of the diagonal band of Broca, and the stria terminalis. In the periphery, NO synthase occurs in the endothelium of blood vessels and, most prominently, in autonomic nerve plexuses. For instance, the enzyme is concentrated in the nerve plexus of the retina, which innervates the choroid and retinal pigmented epithelium, and sympathetic fibers in the adrenal medulla. We have not detected NO synthase in macrophages, perhaps because the macrophage enzyme, whose co-factor requirements differ from the brain and endothelial enzyme, does not react with our antiserum. Messenger molecules - potential for selectivity and plasticity If one takes into consideration second messenger and intercellular messenger molecules, enzyme and receptor subtypes, as well as neurotransmitters, the resultant 'wiring diagram' of the cerebellum is considerably enriched in potential selectivity of signal communication. Both parallel and climbing fibers excite Purkinje cells via direct ionic mechanisms as well as second messenger effects. Both climbing and parallel fibers activate Purkinje cells with complex patterns of increases in the permeability of Ca2+ and sodium which may involve Ca~+-dependent dendritic spikes 7,8. Differential roles of ionotropic and second messenger-linked receptors in synaptic communication in the cerebellum are still uncertain. Electrophysiological responses in Purkinje cells to stimulation of parallel fibers are blocked by the glutamate antagonist kynurenate, and the quisqualate antagonist CNQX n, indicating a role for the quisqualate subtype of ionotropic receptor and ruling out a direct role for the Quis-PI receptor, which is unaffected by CNQX 1a,7°. The two forms of response to parallel fiber activation may have different functions, with PI turnover conveying trophic or other information. Aspartic acid, which may mediate climbing fiber TINS, Vol. 13, No. 6, 1990
effects, is antagonized by APV (aminophosphonovalerate), suggesting an NMDA-related receptorlL Given the selectivity implicit in the subtypes of ionotropic glutamate responses and the dendritic spatial complexity available for different cerebellar afferents, why is there such a plethora of second messengers as well? Second messenger systems generally exert slower, more sustained effects than ionotropic systems 71. Second messengers provide more opportunities for convergence and divergence between different receptors and ion channels 71. Further, an abundance of molecular entities, each with a specific distribution, affords a greater potential for selective regulation. Thus, the PI cycle involves a receptor coupled to a G protein, which activates phospholipase C (PLC), generating two second messengers, one mobilizing Ca2+ stores, the other activating protein kinase C 73 (Fig. 2). Messenger RNA and protein for isozymes of PLC have very different distributions in brain, with one isozyme (PLC~) highly localized to Purkinje cells in cerebellum, while others are present at lower levels in both Purkinje cells and granule cells 72-74. Isozymes of PKC also have different distributions 75. Each of these molecules may be regulated by different mechanisms. The differential distribution of GTP-binding protein G~ isoforms and their mRNAs has provided clues to their functions 76,77. Gs and Gi mediate, respectively, stimulation and inhibition of adenylate cyclaseTM. However, the identity of the G protein associated with stimulation of PI turnover has until recently been uncertain. Based on a similarity between the distributions of Go and IP3 receptors, we suggested that Go might activate the PI cycle in brain 76,77,79. Recently, Moriarty et al. 80 have provided direct evidence that Go is responsible for the PI-mediated electrophysiological response to acetylcholine in Xenopus oocytes. They found that purified Go reconstitutes the response to acetylcholine in pertussis toxin-treated oocytes, and that G,o subunits activated by GTP-¥-S directly evoked the electrophysiological response. There is a greater potential for interactions and crosstalk among different afferents where there are more molecular nodal points. A link between cAMP and PI second messenger systems in Purkinje cells is suggested by our observations that the IP3 receptor can be phosphorylated by cAMP-dependent protein kinase 81 (see Fig. 2). This phosphorylation does not markedly alter the affinity of IP3 for its binding sites; however, it renders IP3 only one-tenth as potent in releasing Ca2+. Additionally, cAMP-dependent phosphorylation of the Ca2+ pump in Purkinje cells enhances the uptake of 45Ca2+ into Purkinje cells in brain sections, so that IP3 acts upon a pool of 45Ca2+ about three times larger than in the unphosphorylated state. Thus, the interaction between cAMP and the IP3 receptor under physiological circumstances may be complex. Finally, interactions among the messenger molecules described above may have a role in synaptic plasticity. In the cerebellum, long-term depression (LTD) is in some respects analogous to long-term potentiation (LTP) in the hippocampus. In the CA1 region of the hippocampus the conjunction of two specific synaptic inputs to a pyramidal cell strengthens one of them, so that its subsequent input to that pyramidal cell is potentiated, leading to greater TINS, VoL 13, No. 6, 1990
excitation of the neuron to which the pyramidal cell projects. In the cerebellum, the conjunction of parallel fiber and climbing fiber stimulation leads to depression of the ability of the parallel fiber to activate the Purkinje cells2. Since the output of the Purkinje cells is inhibitory, the net effect, as in the hippocampus, will be increased firing of the Purkinje cell's target neuron. The receptor at the parallel fiber synapse in LTD is of the quisqualate ionotropic or metabotropic type 83, unlike hippocampal LTP in CA1 and dentate gyrus, which involves the NMDA receptor. One might predict that LTD would involve decreased ability of the parallel fiber to stimulate PI turnover. In the hippocampus, there would appear to be two phases to LTP, the first presynapfic, the second postsynaptic 84. This has led to the hypothesis that trophic factors or neuromodulators transfer information during the course of LTP, first from the postsynaptic cell to the presynaptic terminal, then from the presynaptic terminal back again s5. Williams and Bfiss86 have suggested that arachidonic acid acts as a retrograde messenger from the postsynaptic induction site to the presynaptic terminal in hippocampus; perhaps it also has a role in LTD in the cerebellum. Other modulators, such as NO, adenosine or taurine, could also be involved. Long-term plastic changes in neurons probably involve alterations in transcription of specific genes, as have been described for growth factors, acting via various second messenger systems 87-s9. Related actions are mediated by cellular oncogenes, several of which are present in high concentrations in brain 9°,91. Several oncogenes are selectively expressed in particular cells in cerebellum: H-ras protein is localized in Purkinje cells of adult rats, as well as in the cerebral cortex 92. Purkinje cells of adult chicken contain more pp62c-yes than any other cells in brain 9°. Cerebellar transcripts of c-myc can similarly be localized almost entirely to Purkinje cells 93. Neurotransmitter receptors, such as the serotonin 1C receptor, can, under certain circumstances, be oncogenes 94. As noted above, endothelin can be mitogenic for glial cells. The previously sharp boundaries between neurotransmitters, neuromodulators and trophic factors are thus becoming increasingly blurred. The cerebellum, with its defined synaptic connections, should remain an excellent model to help unravel these complex molecular interactions.
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Acknowledgements Supported by a grant of the International Life SciencesInstitute, Public Health Service Grants MH- 18501, DA-00266, Research ScientistAward DA00074 to SHS, MH43040 to CAR, Training Grant GM07309 to DSB and a
gift from BristolMyers Squibb Company. CAR is a Pew Scholar in the Biomedical Sciences.
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