In vitro studies using cerebellar slices or cultures, as well as in vivo studies have demonstrated that NO formation and release occur in the cerebellar cortex in response to NMDA receptor activation. We have found using intracerebellar microdialysis in awake behaving animals, that this is associated with a large increase in extracellular cGMP levels in the cerebellar cortex. A similar increase in cGMP efflux is seen in response to activation of AMPA or metabotropic glutamate receptors, or to activation of the climbing fiber input. The increase in extracellular cGMP required the Ca2+-dependent activation of NO synthase, and was potentiated by inhibition of phosphodiesterases or organic anion transport. These results suggest a possible role for cGMP as an intercellular messenger in the cerebellar cortex.
In summary, stimuli which elevate Ca2+ levels in granule and/or basket cells will activate NO synthase, which by binding to its receptor, soluble guanylyl cyclase, causes an increase in cGMP. cGMP may then act through protein kinases, phosphodiesterases, ion channels or receptors to affect cerebellar function.
The discovery that the increase in cGMP seen in cerebellar neurons in response to NMDA receptor activation resulted from the release of a diffusible messenger with properties similar to those of the endothelium-derived relaxing factor (EDRF), stimulated great interest in the role of this novel signalling pathway in the nervous system (Garthwaite et al., 1988). With the identification of EDRF as nitric oxide (NO), the enzyme responsible for its synthesis, nitric oxide synthase (NOS) was soon described in brain (Bredt & Snyder, 1990; Knowles et al., 1989; Mayer et al., 1990). Although the NO signalling pathway has now been described in many regions of the central and peripheral nervous systems, the cerebellum remains a key area for researchers interested in the functional significance of this novel messenger molecule.
In the rat, the cerebellum has about double the NOS activity found in other brain regions (Bredt et al., 1991; F"rstermann et al., 1990). With the purification of neuronal NOS, immunohistochemical studies indicated that it was expressed by basket and granule cells and their processes but not by Purkinje cells or glial elements (Bredt et al., 1990). Similar observations were obtained using the NADPH-diaphorase reaction which detects NOS histochemically (Hope et al., 1991; Southam et al., 1992; Vincent & Hope, 1992; Vincent and Kimura, 1992). Likewise, NOS mRNA is highly expressed in the granule cell layer, but is absent from the Purkinje cells (Bredt et al., 1991). The climbing fibers do not appear to contain NOS, since in numerous species, the inferior olive is unstained with NADPH-diaphorase histochemistry (Vincent and Kimura, 1992; Mizukawa et al., 1989; Kowall and Meuller, 1988), and 3-acetylpyridine lesions of the inferior olive have no effect on cerebellar NOS activity (Ikeda et al., 1993). The deep cerebellar nuclei and their mossy fiber afferents to the cerebellar cortex also do not contain NOS-positive neurons.
There have been reports that Purkinje cells do stain weakly for NADPH-diaphorase during early postnatal development (Brning, 1993; Yan et al., 1993). As noted by Brning (1993) this weak staining should be interpreted with caution. It may be the result of poorer fixation of these neonatal brains, since the relationship of NADPH-diaphorase to NO synthase is only apparent following formaldehyde fixation (Hope et al., 1991; Matsumoto et al., 1993). This might also explain a report of NAPDH-diaphorase staining in the rat inferior olive (Southam and Garthwaite, 1993). Indeed, we have seen weak staining of inferior olive or CA1 pyramidal neurons in material that has been poorly fixed (unpublished observations).
When NO is produced from arginine via NOS, the amino acid citrulline is a co-product. Thus citrulline production would be predicted to occur in the NOS-containing basket and granule cells in the cerebellum. However, the enzymes needed to catalyse the conversion of citrulline back into the NO precursor arginine, argininosuccinate synthetase and lysase, are absent from these cells (Arnt-Ramos et al., 1992; Nakamura et al., 1990; 1991). They are instead expressed in a discrete population of neurons just beneath the Purkinje cell layer, which might correspond with Lugaro cells or perhaps the recently described candelabrum cell (Lain and Axelrad, 1994). This suggests that a transcellular metabolic pathway for citrulline and arginine metabolism occurs in the cerebellum. Indeed, a delayed release of arginine has been noted following stimulation of rat cerebellar slices (Hansel et al., 1992).
The cerebellum has relatively low levels of soluble guanylyl cyclase (Greenberg et al., 1978; Hofmann et al., 1977; Nakazawa and Sano, 1974). A number of biochemical studies have sought to localize soluble guanylyl cyclase in the cerebellar cortex. Kainic acid lesions, which kill Purkinje, basket, stellate and Golgi neurons, but largely spare granule cells and glia, result in an almost complete loss of guanylyl cyclase activity and cGMP levels in the cerebellar cortex (Biggio et al., 1978). In contrast, lesions of the germinal layer of the developing cerebellum, which deplete the cerebellum of granule cells, result in an increase in guanylyl cyclase activity (Bunn et al., 1986). Also, the NO donor, sodium nitroprusside, dramatically elevates cGMP levels in granule cell depleted mice (Wood et al., 1994). Thus granule cells do not appear to express significant soluble guanylyl cyclase activity in vivo, although NO-dependent cGMP formation has been well described in cultured granule cells (Kiedrowski et al., 1992; Novelli & Henneberry, 1987).
Studies have also made use of various mutant mice exhibiting cerebellar degeneration to examine the localization of the NO/cGMP system. Nervous, mutant mice, which lack most Purkinje cells, were reported to have a corresponding loss of guanylyl cyclase and cGMP (Mao et al., 1975; Schmidt and Nadi, 1977). Nervous and Purkinje cell degeneration mutant mice also showed a loss of NOS activity in the cerebellar cortex (Ikeda et al., 1993). However, another group has found no change in cerebellar cGMP levels or the response to harmaline in these mutant mice (Wood et al., 1994). Together, these confusing results indicate that secondary changes in enzyme expression occuring during development in these mutant mice might contribute to the observed neurochemical effects.
Immunohistochemical and in situ hybridization observations are largely consistent with the localization of guanylyl cyclase determined biochemically. The mRNAs for the a1 and B1 subunits of soluble guanylyl cyclase are highly expressed in Purkinje cells, with moderate levels seen in stellate, basket and Golgi cells and low levels of expression in granule cells (Furuyama et al., 1993; Matsuoka et al., 1992; Verma et al., 1993). Immunohistochemical studies with monoclonal antibodies to this enzyme have demonstrated strong staining of Purkinje cells, with weak staining of neurons in the molecular and granule cell layers (Ariano et al., 1982; Nakane et al., 1983), although earlier studies with a polyclonal serum that was not well characterized resulted in staining of all cerebellar cells (Zwiller et al., 1981).
In agreement with these studies, other biochemical and immunohistochemical observations have noted that cGMP-dependent protein kinase is highly expressed in Purkinje cells (Bandle & Guidotti, 1976; DeCamilli et al. 1984; Dolphin et al., 1983; Lohmann et al. 1981, Nairn and Greengard, 1983). Furthermore, a substrate for this kinase, termed G-substrate, is also concentrated in these cells (Detre et al., 1984; Schlichter et al., 1978; 1980).
Although a calmodulin-dependent cyclic nucleotide phosphodiesterase is present in Purkinje cells, where its expression is regulated by the climbing fiber input (Balaban et al., 1989), the activity of cyclic nucleotide phosphodiesterase, measured either in the presence or absence of calmodulin is the lowest of any brain region (Greenberg et al., 1978; Hofmann et al., 1977). This might account for the fact that cGMP levels in the cerebellum are much higher than those in other brain regions, and increase dramatically in response to activation.
Together, these anatomical studies suggest a model in which NO is produced by the calcium- dependent NOS present in basket and granule cells, and acts on its receptor, the soluble guanylyl cyclase, which is primarily localized in the Purkinje cells, as well as in inhibitory interneurons. However, pharmacological studies have provided results that make this relatively simple senario controversial. In particular, immunohistochemical studies have been undertaken to localize those cells responding to NO with an increase in cGMP. A dramatic increase in cGMP-immunoreactivity in Bergmann glia, granule cells and glomeruli (De Vente et al., 1989; 1990; de Vente and Steinbusch, 1992; Southam et al., 1992) and moderate staining of Purkinje cell bodies (Southam and Garthwaite, 1993) has been seen following sodium nitroprusside. Some cGMP staining of stellate and basket cells has also been described in unstimulated cerebellum, but Purkinje cells appeared to be unstained (Chan- Palay and Palay, 1979). However, other immunohistochemical studies using glutaraldehyde fixation and high-affinity monoclonal antibodies have revealed intense cGMP-immunoreactivity in Purkinje cells (Sakaue et al., 1988). A possible explanation for these results might be provided by the discovery that NO induces a large increase in extracellular cGMP, which is then cleared from the extracellular space by a probenecid-sensitive mechanism (Luo et al., 1994; Tj"rnhammer et al., 1986; Vallebuona and Raiteri, 1993). Thus cGMP might be produced primarily in Purkinje cells, but rapidly leaves these cells to be accumulated by the Bergmann glia.
NO and the Regulation of Cerebellar cGMP Levels
Many studies using a wide variety of techniques are consistant with there being a tonic production of NO and cGMP in the cerebellar cortex. The levels of cGMP in the cerebellum are an order of magnitude greater than those in other brain regions. cGMP levels can be decreased by elevating cerebellar GABA transmission (Mao et al. 1974a,b; 1975; Dodson and Johnson, 1980), and increased dramatically by blocking GABA action (Biggio et al. 1977a; Mailman et al., 1978; Mao et al., 1974a,b). Conversely, activation of excitatory amino acid receptors increases cGMP levels in the cerebellar cortex (Briley et al., 1979; Danysz et al., 1989; Ferrendelli et al., 1974; Mao et al., 1974a; Wood et al., 1982). Furthermore, activation of the climbing fiber input using harmaline also produces large increases in cerebellar cGMP levels (Biggio and Guidotti, 1976; Biggio et al., 1977b; Cross et al., 1993; Guidotti et al., 1975; Luo et al., 1994; Mao et al., 1974a).
How does climbing fiber activation regulate cerebellar NO and cGMP production? Climbing fibers activate Purkinje cells via an AMPA type receptor, which can be blocked by CNQX, and mature Purkinje cells appear to lack NMDA receptors (Farrant and Cull-Candy, 1991) and do not express NOS (Bredt et al., 1990; Southam et al., 1992; Vincent & Hope, 1992; Vincent & Kimura, 1992). Since the harmaline-induced increase in cerebellar cGMP can be blocked by NMDA receptor antagonists (Luo et al., 1994; Wood et al., 1982; 1990), the climbing fiber input to the Purkinje cells would not appear to be responsible. The NOS-containing granule and/or basket cells appear to be required for climbing fiber- induced increases in cerebellar cGMP (Wood et al., 1994), however, they do not appear to receive a substantial direct climbing fiber input (Leranth and Hamori, 1981). This suggests that an indirect pathway may be involved. The inferior olive does innervate the deep cerebellar nuclei (Audinat et al., 1992; Llinas and Muhlethaler, 1988), which in turn give rise to the major mossy fiber input to the granule cells, activating these neurons via both NMDA and AMPA receptors (Garthwaite and Brodbelt, 1989). Thus harmaline-induced stimulation of the inferior olive could result in excitation of the mossy fiber input to the granule cells from the deep cerebellar nuclei, and this might account for the activation of NOS seen following climbing fiber activation (Shibuki, 1990).
The increase in cerebellar cGMP in response to excitatory amino acid receptor agonists or harmaline is mediated by activation of NOS, since it is effectively blocked by a variety of NOS inhibitors (Bansinath et al., 1993; East and Garthwaite, 1990; Garthwaite, 1991; Garthwaite et al., 1989a; Luo et al., 1994; Southam et al., 1991; Wood et al., 1990). Various NO donors can themselves elevate cerebellar cGMP levels (Southam and Garthwaite, 1991a). Furthermore, the increase in cGMP seen in cerebellar slices in response to glutamate receptor activation is inhibited by hemoglobin, suggesting that NO must travel between cells to affect guanylyl cyclase (Southam and Garthwaite, 1991b).
Electrochemical methods have been used to demonstrate NO release from adult rat cerebellar slice preparations following electrical stimulation (Shibuki, 1990; Shibuki and Okada, 1991). Furthermore, NO release from cerebellar slices following depolarization or NMDA receptor activation has been examined using a chemiluminescence assay (Dickie et al., 1990; 1992). NMDA receptor activation has also been shown to elevate NO release in the cerebellar cortex in vivo in awake, unrestrained animals (Luo et al., 1993). This study also provided evidence for a tonic NMDA-induced NO production in the intact cerebellar cortex.
In the cerebellar cortex, the NMDA induced activation of NOS has been suggested to result largely from direct activation of NMDA receptors on the neurons containing NOS, since the increase in cGMP seen in response to NMDA is largely unaffected by tetrodotoxin (Luo et al., 1994; Southam et al., 1991). However, the recently characterized presynaptic NMDA receptor could also be involved (Smirnova et al., 1993). Indeed, some in vivo experiments imply a role for monoaminergic innervation in mediating the increase in cGMP seen in response to NMDA receptor activation. The increases in cerebellar cGMP levels seen in response to harmaline or local NMDA activation was blocked by prior reserpinization (Wood et al., 1992). Reserpine treatment also decreased basal cGMP levels by about 50% (Burkard et al., 1976; Ferrendelli et al., 1972, Wood et al., 1992). Pharmacological studies indicate that the stimulatory effect of harmaline or NMDA receptor activation was blocked by adrenergic antagonists of the a1A type acting within the cerebellar cortex (Rao et al., 1991). Consistent with this are the observations that a1 antagonists themselves can decrease, while a1 agonists or noradrenaline itself can increase cerebellar cGMP levels (Haidamous et al., 1980). However, in some in vitro studies using slices from neonatal rat cerebellum, noradrenaline antagonized the cGMP increase induced by NMDA (Carter et al., 1988). Biochemical and electrophysiological experiments indicate that phencyclidine-sensitive NMDA receptors increase noradrenaline release in the cerebellum (Marwaha et al., 1980; Yi et al., 1988). This suggests that NMDA receptors on noradrenergic nerve endings in the cerebellum may, by stimulating noradrenaline release, affect cGMP levels via an a1-receptor mechanism.
Although most attention has been paid to the role of NMDA receptors in the stimulation of NOS and the production of cGMP, activation of AMPA, kainate or metabotropic glutamate receptors also produces an increase in cGMP, again apparently via a direct action on NOS-containing cerebellar neurons (Garthwaite et al., 1989b; Luo et al., 1994; Okada, 1992; Southam et al., 1991). The cGMP increase seen in response to AMPA is of similar magnitude to the NMDA-induced increase. In contrast, the cGMP increase seen in response to metabotropic receptor activation is considerably smaller. This suggests that releasing calcium from intracellular stores, which appears to mediate metabotropic receptor-induced NO production (Okada, 1992), is much less effective in activating cerebellar NOS, than is activation of the ionotropic glutamate receptors. Both the cerebellar basket cells (G"rcs et al., 1993) and granule cells are known to possess metabotropic glutamate receptors (Nicoletti et al., 1986; Fagni et al., 1991; East and Garthwaite, 1992), some of which may be present on the terminals making contact with Purkinje cells (Glaum et al., 1992; Takagi et al., 1992).
In addition to the excitatory amino acids, a variety of other neurotransmitter systems can affect cerebellar NO and cGMP production. Thus manipulations of cholinergic (Dinnendahl and Stock, 1975; Dodson and Johnson, 1979), dopaminergic (Biggio et al., 1977; Breese et al., 1978; Burkard et al., 1976; Ferrendelli et al., 1972) and opioid (Biggio et al., 1977) systems outside the cerebellum are known to affect cerebellar cGMP levels. These actions appear to be mediated by activation of the mossy fiber afferents to the cerebellar cortex, which in turn leads to stimulation of the NOS-containing granule and/or basket cells (Wood, 1991).
NO, cGMP and Cerebellar Synaptic Plasticity
When EDRF was first suggested as an intercellular messenger in the cerebellum, its unique properties and potential significance for synaptic plasticity were noted (Garthwaite et al., 1988). The inhibition of vestibuloocular reflex adaptation by subdural hemoglobin suggested a possible role for NO in synaptic plasticity in this area (Nagao and Ito, 1991). There have been some reports of NO-catalyzed ADP-ribosylation of proteins being a possible action of NO in the brain (Brne & Lapetina, 1989). However, it now appears that NO does not catalyze such a reaction, rather, through S-nitrosylation, covalent binding of NAD to some proteins can occur (McDonald and Moss, 1993; Zhang & Snyder, 1992). S-nitrosylation of proteins may be of great importance in the neurotoxic actions of NO, but is unlikely to be involved in the normal intercellular signalling by this molecule. Instead, NO appeaers to act by mediating agonist-induced increases in cGMP.
Data consistent with a role for the NO-cGMP signal transduction system in the long term depression (LTD) of AMPA responses in Purkinje cells have been obtained by various groups. Ito and Karachot (1990) demonstrated that the desensitization of Purkinje cell responses to AMPA in rat cerebellar slices was abolished by Ca2+ chelators, hemoglobin, the nitric oxide synthase inhibitor NG- nitroarginine, and an inhibitor of cGMP-dependent protein kinase. Experiments on month old rat cerebellar slices demonstrated an LTD of parallel fiber mediated EPSPs when they were paired with depolarization-induced calcium spikes in Purkinje cells (Crepel and Jaillard, 1990). This depression was prevented by bath application of polymyxin B, L-NG-monomethylarginine or methylene blue, again suggesting a role for protein kinase C, NO and cGMP. Furthermore, co-application of sodium nitroprusside or 8-bromo-cGMP with AMPA or parallel fiber stimulation could induce LTD, suggesting that elevated cGMP could substitute for climbing fiber excitation of the Purkinje cells (Ito and Karachot, 1990; Shibuki and Okada, 1991; 1992). However, the parallel fibers contain NOS and would be an endogenous source of NO, while the climbing fibers lack NOS. Thus why NO donors should substitute for climbing fiber activation is not apparent.
In thin slices from two-week old rat cerebellum, whole-cell, patch clamp experiments demonstrated that climbing fiber activation or direct depolarization of Purkinje cells, delivered in conjunction with parallel fiber activation, induced an LTD of the parallel-fiber synapse (Daniel et al., 1993; Konnerth et al. 1992). The depolarization-induced rise in intracellular calcium in the Purkinje cell was both necessary and sufficient to initiate LTD (Konnerth et al., 1992). Thus induction of LTD appears dependent upon a calcium-mediated event occuring within the Purkinje cells coincident with glutamate receptor activation. The calmodulin-dependent activation of NO synthase would not appear responsible, since Purkinje cells do not express NOS (Bredt et al., 1990; Southam et al., 1992; Vincent & Kimura, 1992). Indeed, Daniel et al. (1993) demonstrated that while bath application of L-NG- monomethylarginine reduced the amplitude of LTD in an L-arginine reversible manner, inclusion of this NOS inhibitor in the Purkinje cell recording pipette did not prevent LTD induction. This provides strong evidence against a role of NOS within Purkinje cells in this phenomenon. Likewise, a direct Ca2+-dependent effect on guanylyl cyclase in the Purkinje cells appears unlikely, since this enzyme is unaffected by Ca2+ (Mayer et al., 1992).
Experiments using the thin-slice method also yield controversial results regarding the action of NO donors on LTD. Daniel et al., (1993) found that at 8 mM, sodium nitroprusside inhibited the parallel fibre-mediated EPSP when applied for 4-9 min. A similar result was observed with 3 mM SIN- 1. However, others found that sodium nitroprusside applied at 3 mM for up to 15 min. had no effect on the membrane properties of Purkinje cells, nor on the amplitude of the parallel fiber induced EPSP (Glaum et al., 1992). Likewise, in perforated-patch recordings from cultured rat Purkinje cells, 3 mM sodium nitroprusside had no effect on the response to iontophoretic glutamate (Linden and Connor 1992). The slight differences in drug concentrations in these studies is unlikely to offer an explanation for these discrepancies, since the massive increase in cGMP levels produced would have been similar (Southam and Garthwaite, 1991a). Indeed, these doses of sodium nitroprusside would likely be toxic to the neurons in these preparations (Dawson et al., 1991).
Recent studies have used NADPH-diaphorase to localize NO synthase in the chicken cerebellum. As in the rat, the granule cells and some cells in the molecular layer were stained, but the Purkinje cells and the neurons of the cerebellar nuclei were unstained (Brning, 1993). Studies undertaken in cultured chick Purkinje cells demonstrated that the response to iontophoretic AMPA, monitored by intracellular or whole-cell recording, was suppressed by trans-ACPD, a metabotropic glutamate receptor agonist, and this suppression could be blocked by hemoglobin, the NOS inhibitor L- NG-monomethylarginine, or the cGMP-dependent protein kinase inhibitor KT5823, or mimicked by the NO donors sodium nitroprusside and 3-morpholinosydnonimine (Mori-Okamoto et al., 1993). However, potassium ferricyanide was also able to mimic the suppression, via a cGMP-independent mechanism. Furthermore, the trans-ACPD effect could also be prevented by blocking protein kinase C activity. Similar observations were made using slice preparations from adult rat cerebellum (Ito and Karachot 1990), although studies with cultured rat Purkinje cells found that preincubation with nitroarginine or hemoglobin for 25 minutes did not attenuate the depression of glutamate responses induced by glutamate/depolarization pairing (Linden & Connor, 1992).
Most of these studies have made use of non-specific NO donors, or NOS inhibitors which are non-selective, and can affect numerous other cellular processes (Peterson et al., 1992; Schmidt et al., 1993). However, together, these studies are consistent with the idea that while not necessary for the induction of LTD, stimulation of basket and/or granule cells, leads to NOS activation, and the NO so formed then diffuses into the Purkinje cells were it activates soluble guanylyl cyclase. The resulting elevation in cGMP levels leads to activation of cGMP-dependent protein kinase, which through a phosphorylation cascade, can depress the AMPA response. Direct phosphorylation of the AMPA receptor by cGMP-dependent protein kinase would appear unlikely, since the GluR2 and GluR3 receptor subtypes present in Purkinje cells (Petralia and Wenthold 1992) lack a consensus sequence for this kinase (Boulter et al., 1990; Sommer et al. 1990; Kennelly and Krebs 1991). Direct phosphorylation of these receptors by protein kinase C does appear to be a possibility (Kennelly and Krebs 1991). It may be that cGMP-dependent protein kinase, by phosphorylating G-substrate, inhibits protein phosphatases that would antagonize this effect of protein kinase C. Perhaps when direct depolarization of the Purkinje cells is used to induce LTD (Linden and Connor 1992; Konnerth et al. 1992), the resultant increase in intracellular calcium, and protein kinase C activity is sufficient to suppress AMPA responses in the absence of NO-dependent, cGMP-induced activation of cGMP-dependent protein kinase. In this regard, it is important to note that trans-ACPD alone does not elevate somatic calcium concentration unless the Purkinje cell fires calcium-dependent action potentials (Glaum et al. 1992).
Other Roles for cerebellar NO and cGMP
cAMP-dependent protein kinase phosphorylation of CREB and related transcription factors allows the cAMP signal transduction pathway to regulate gene expression in target neurons. By analogy, NO-dependent cGMP production might influence gene expression in neurons such as the Purkinje cells, which express both soluble guanylyl cyclase and cGMP-dependent protein kinase. Recent work on PC12 cells indicates that NO can induce immediate early gene expression via the activation of cGMP-dependent protein kinase (Haby et al., 1994). NMDA receptor activation can induce c-fos expression in cultured cerebellar granule cells (Szekely et al., 1989). However, this appears to be mediated by protein kinase C, and dibutyryl cGMP did not increase c-fos expression. Likewise, evidence for a cGMP response element regulating gene expression in Purkinje cells or other neurons, is lacking.
Other substrates for the cGMP-dependent protein kinase in the cerebellum might include the ryanodine receptor, which is highly expressed in Purkinje cells (Sharp et al., 1993), and has been shown to be phosphorylated by this kinase (Suko et al., 1993), causing an enhancement of its calcium releasing activity (Herrmann-Frank & Varsanyi, 1993). Furthermore, cGMP might enhance the production of the proposed endogenous ligand for this receptor, cyclic ADP-ribose (Galione et al., 1993). The IP3 receptor, which is also highly expressed in Purkinje cells (Sharp et al., 1993) can also be phosphorylated in vitro by the cGMP-dependent protein kinase (Lincoln & Cornwell, 1993).
A particularly exciting recent discovery is the demonstration that the gene for a cyclic nucleotide-gated channel is expressed in rabbit cerebellum (Biel et al., 1993). This channel has properties similar to those of the cation channel in the olfactory epithelium, opening in response to an increase in intracellular cGMP. It will be of great importance to determine which cells in the cerebellum express this channel, since one would predict that a NO-induced increase in intracellular cGMP might depolarize cells expressing such a channel.
Finally, given the extraordinarily high levels of cGMP in the cerebellar cortex, and the demonstration of a NOS-dependent increase in extracellular cGMP following stimulation, a role for this cyclic nucleotide in intercellular communication might also be considered (Luo et al., 1994). Electrophysiological effects of extracellular cGMP have been reported in the cerebellum (Hoffer et al., 1971; Siggins et al., 1976). Thus cell surface, G protein-linked receptors similar to those described for cAMP (Klein et al., 1988; Sorbera & Morad, 1991) may exist for cGMP.
Since its discovery as a signal transduction molecule in the cerebellum, intense interest has been focused on the role of NO. It is now apparent that stimuli which increase the intracellular free calcium within basket and granule cells can lead to the calmodulin-dependent activation of NOS. The NO produced will diffuse to reach and activate its target, soluble guanylyl cyclase, within Purkinje cells, and perhaps other cell types. It is clear from the biochemical neuroanatomy of this system that while NO may be involved in synaptic plasticity, it does not appear to be essential for the induction of LTD. The function of NO is to activate soluble guanylyl cyclase. Thus to understand the functional significance of this novel messenger molecule, it will be necessary to elucidate the roles of cGMP in cerebellar physiology.
The work from my laboratory on NO and cGMP was supported by a grant from the Medical Research Council of Canada.
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Figure Caption
[Note: Figures avaiable only in hard copy version]
Fig. 1. Schematic diagram illustrating the neuronal mechanisms involved in synaptic activity in the cerebellar cortex. Long-term depression (LTD) of the parallel fiber to Purkinje cell AMPA response requires coincident climbing fiber and parallel fiber activation.
The massive climbing fiber synapse, acting on AMPA receptors, can result in sufficient depolarization to activate P-type calcium channels in the Purkinje cell dendrites. This calcium increase may be further amplified through interaction with the calcium-dependent calcium release system (ryanodine receptor) present in Purkinje cell dendrites.
The granule cells, which give rise to the parallel fibers, can be activated by mossy fibers via both AMPA and NMDA receptors. Activation of granule cells results in the calcium-dependent formation of NO within these neurons. Such NO formation in the parallel fiber nerve terminals could thus act on Purkinje cell dendritic spines receiving the parallel fiber input. The parallel fiber induced EPSP in Purkinje cells is mediated by an AMPA receptor, although a metabotropic glutamate receptor can also be activated, resulting in the generation of IP3 and diacyl glycerol (DAG) and activation of protein kinase C (PKC) within the Purkinje cell dendritic spines. Coactivation of both the AMPA and metabotropic receptors and subsequent PKC activation appear necessary for the induction of LTD of the AMPA response.
Within the Purkinje cell, NO appears to activate soluble guanylyl cyclase (GC), which, via generation of cGMP, leads to activation of cGMP-dependent protein kinase (PKG). PKG could possibly influence LTD by various mechanisms including actions on the two intracellular calcium release systems, or phosphorylation of other targets such as protein phosphatase inhibitors.