The Cerebellum

, Volume 11, Issue 1, pp 50–61

Role of Nitric Oxide in Cerebellar Development and Function: Focus on Granule Neurons

Authors

    • Department of BiologyUniversity of Bologna
Article

DOI: 10.1007/s12311-010-0234-1

Cite this article as:
Contestabile, A. Cerebellum (2012) 11: 50. doi:10.1007/s12311-010-0234-1

Abstract

More than 20 years of research have firmly established important roles of the diffusible messenger molecule, nitric oxide (NO), in cerebellar development and function. Granule neurons are main players in every NO-related mechanism involving cerebellar function and dysfunction. Granule neurons are endowed with remarkable amounts of the Ca2+-dependent neuronal isoform of nitric oxide synthase and can directly respond to endogenously produced NO or induce responses in neighboring cells taking advantage of the high diffusibility of the molecule. Nitric oxide acts as a negative regulator of granule cell precursor proliferation and promotes survival and differentiation of these neurons. Nitric oxide is neuroprotective towards granule neurons challenged with toxic insults. Nitric oxide is a main regulator of bidirectional plasticity at parallel fiber-Purkinje neuron synapses, inducing long-term depression (LTD) or long-term potentiation (LTP) depending on postsynaptic Ca2+ levels, thus playing a central role in cerebellar learning related to motor control. Granule neurons cooperate with glial cells, in particular with microglia, in the regulation of NO production through the respective forms of NOS present in the two cellular types. Aim of the present paper is to review the state of the art and the improvement of our understanding of NO functions in cerebellar granule neurons obtained during the last two decades and to outline possible future development of the research.

Keywords

Nitric oxidecGMP signalingCerebellar granule neuronsNeurogenesisSurvival and differentiationSynaptic plasticityGlial cell interactions

Introduction

More than 20 years ago, the endothelium-derived relaxing factor of blood vessel smooth muscles was identified as nitric oxide (NO), thus starting the long story of amazing discoveries on the role of this simple gaseous molecule in vascular physiology [14]. At about the same time, a parallel story started to be told regarding NO as a signaling molecule in the brain, with the discovery that a diffusible messenger, identified as NO and eliciting a cGMP surge, was produced and released in response to glutamate acting at N-methyl-d-aspartate (NMDA) synaptic receptor [5]. It is noteworthy and relevant to underline the importance for neuroscience research of the neuronal population to which this special issue of the journal Cerebellum is dedicated that this milestone discovery was made in cerebellar granule neurons. The extent of research dealing with the physiologic and pathologic roles of NO in nervous tissue is impressive and has been repeatedly reviewed during the years [611]. Among many remarkable discoveries, this research effort has produced evidence for involvement of NO in such diverse functions as: neuronal proliferation, survival and differentiation; synaptic transmission and plasticity; learning and memory; regulation of circadian rhythms. Furthermore, deregulation of NO production is involved in several neural pathologies from neurodegenerative diseases to tumors arising from neural cells.

One important issue when dealing with such an easily diffusible and labile molecule as NO is to understand how its functions comply with the physicochemical constraints governing its actual concentration at various times and spaces from the moment and the place of production. The reader is referred to some papers of Garthwaite and collaborators who have masterly reviewed recent and very interesting developments on these issues [1114]. The single most important conceptual novelty raised by these studies is that NO exerts its physiological actions at concentrations (0.1–5 nM) orders of magnitude lower than those previously proposed [14]. At these very low concentrations, half-life of produced NO may not exceed few tents of milliseconds, as evaluated by experiments in cerebellar slices [13], again a value much lower than previously supposed. However, the physiologic relevance of these very low concentrations of such a labile molecule is ensured by its high diffusion rate (some micrometer per millisecond and up to 8 μm/ms according to some calculations) and by the very efficient coupling with NO receptor activation, exemplified by the higher than thousand-fold amplification of NO signaling in terms of cGMP produced by guanylyl cyclase [13, 15]. Regarding specifically brain activities of NO, it is noteworthy to remember that the efficiency of its synaptic action is strongly enhanced by the strict functional coupling with the synaptic receptor machinery, well exemplified by the PDZ domain of NMDA receptors [13, 16, 17].

Aim of the present article is to focus on the role of NO in cerebellar development and function with special emphasis on those aspects in which granule neurons, the largest neuronal population of the cerebellum, are primarily involved from neurogenesis to survival, synaptic function, plasticity, and interaction with other cellular components.

Production of Cerebellar Nitric Oxide, its Targets and Transduction Mechanisms

Among the three enzymatic isoforms producing NO by converting arginine to citrulline, the two constitutive Ca2+-dependent isoforms neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) are expressed in the cerebellum [18, 19], while the inducible isoform (iNOS) is not appreciably expressed in normal animals but starts to appear in pathologic conditions [20, 21]. The nNOS isoform has the distinctively highest level of expression, and its protein content in the cerebellum is comparatively higher than in other brain regions [18, 19, 22]. Cerebellar neuronal populations expressing nNOS are represented by mature granule neurons as well as by the molecular layer interneurons, basket and stellate cells, which gives rise to very strong stain of both the granular and the molecular layers in immunocytochemical preparations [18, 2325]. Regarding granule cells, there are converging evidences that expression of nNOS and production of NO are strongly dependent on differentiation. Expression and activity of nNOS in the developing rat cerebellum grow slowly during the first postnatal week [26], granule neurons show detectable NOS immunoreactivity only after migration in the internal granular layer [25], and measurable Ca2+-dependent NOS activity sharply increases in cerebellar homogenates in parallel with the completion of the migratory process up to the end of the third postnatal week, growing then more smoothly towards adulthood [27]. Quantitative analysis of NOS in the developing rat cerebellum based on freeze-dried microsamples of the various layers is in agreement with a progressive increase during development and also documents that declining activity is present in the external granular layer, and increasing activity takes place in the internal granular layer and in the molecular layer with differentiation [28]. Experiments in vitro, based on the standard methodology of primary cultures of cerebellar granule cells explanted from 7-day-old rat pups [29], were in good agreement with in vivo data regarding the timing of nNOS expression by granule neurons [30]. Levels of nNOS protein detected through western blot peaked after 7–9 days in vitro, when granule neurons in cultures reach their functionally mature state [29] and biochemically measured enzymatic activity behaved in an exactly parallel way [30]. These results have been essentially confirmed by studying in parallel nNOS protein expression and nNOS mRNA synthesis over 2 weeks of culture in cerebellar granule neurons [31]. In granule cell cultures from mouse cerebellum, a similar pattern of nNOS expression and activity was detected, and nitrite accumulation in the medium (a conventional way to measure NO production) increased from 5 to 9 days in vitro, reaching then a plateau [32]. While nNOS expression at the mRNA and protein level show a good degree of parallelism [31], the protein expression and activity can be post-transcriptionally modulated as demonstrated by up-regulation of nNOS caused by heregulin, a member of the family of neuregulins, through MAP kinase pathway in granule cell cultures [33]. The importance of NOS expression and NO production for survival and differentiation of granule neurons will be considered in the following paragraphs.

Main receptor target for NO is represented by the so called “soluble” guanylyl cyclase, an αβ-heterodimer with two possible isoforms, α1β1 and α2β1, whose activities are similarly sensitive to NO concentration [12, 34, 35]. The term soluble applied to the enzyme is currently used in scientific literature but is rather misleading as a conspicuous fraction of the protein is actually recovered from membrane preparations [34, 36]. Immunocytochemical studies in the rat brain demonstrated a widespread distribution of both α and β subunits and in several instances, including the cerebellum, a substantial degree of co-localization with nNOS [37]. The two main heterodimers also display differences in subcellular localization, α1β1 being mainly cytosolic but able to switch to membrane association under various conditions of cellular signaling [38, 39], while α2β1 being enriched in synaptic membranes through binding to post-synaptic density-related proteins [40]. Kinetics of association with and activation by NO of guanylyl cyclase have been extensively characterized both in cell-free systems and in cellular settings [34, 4143]. Activation of guanylyl cyclase by NO results in surge of cellular cGMP which is the main cellular transducer of NO effects and whose levels and time course are downstream regulated by phosphodiesterases. In addition to regulating various channel proteins by direct binding [44, 45], main target of cGMP is represented by protein kinases G (PKGs) which in turn transduce signals to a vast array of other proteins through phosphorylation [4648].

In some instances, non-cGMP-dependent effects of NO are described, implying that other receptors for NO do exist. Nitric oxide, and even more actively its derivative peroxynitrite, can react with several amino acidic protein residues, most notably tyrosine, tryptophan, and cysteine, through reaction of nitration and nitrosylation, or chemically more correctly nitrosation [49], of many proteins [5053]. While this mechanism of modification of protein function may have regulatory importance for cellular functions, its real occurrence in cellular milieu and its actual roles are still controversial and debated [11]. Some recent results [54] have highlighted the importance of NO-mediated protein nitrosylation for the neurotrophin-induced gene expression by the transcription factor cyclic AMP responsive-element-binding protein (CREB), a very important transcription factor for the regulation of many brain functions [55, 56]. Furthermore, NO-mediated epigenetic mechanisms of chromatin remodeling have been recently demonstrated in developing neurons through nitrosylation of histone deacetylases [57, 58]. In addition to the previously demonstrated CREB regulation of neuronal survival through NO/cGMP system [59], and of the recently reviewed regulation of several transcription factors by NO in neurons and neural-derived tumor cells [10], these results highlight a main road for NO transduction signaling in which this labile molecule may have long-lasting effects on target cells.

Another possible interactor is represented by mitochondrial cytochrome c oxidase, where NO may compete for O2 binding inhibiting respiration, an effect that is more likely to occur in pathologic than in physiologic conditions due to the relatively high concentration of NO required [60, 61].

Nitric Oxide in Granule Cell Neurogenesis

Nitric oxide is an important modulator of proliferation in many types of normal and tumor cells [62, 63]. Regarding cells of neural origin, first evidence for negative regulation of proliferation by NO was obtained in PC12 cells 15 years ago [64]. A similar effect in developmental neurogenesis was first reported in insects and non-mammalian vertebrates [6569]. In mammals, NO has been shown to down-regulate adult neurogenesis which takes place lifelong among granule cells of the hippocampal dentate gyrus and neuronal precursors originating from the forebrain subventricular zone and migrating towards the olfactory bulb [7073]. Evidence for negative regulation of cerebellar granule cell precursor proliferation by NO emerged from a culture study of neonatal rat cerebellum slices where NOS inhibition was able to maintain an age-dependent higher proliferation rate among neuronal precursors localized in the external granular layer [74]. This pharmacologic result suggested that NO physiologically acted as a factor down-regulating proliferation of granule cell precursors and favoring their switch towards terminal differentiation. In a series of studies based on primary cultures of dissociated cerebellar granule cells differently enriched in granule cell precursors, we have demonstrated that NOS inhibition increases precursor proliferation relieving the NO/cGMP/PKG inhibition of proliferation [26, 75]. More recently, we were able to directly demonstrate NO-dependent down-regulation of precursor proliferation through 24 h exposure of cultures from neonatal rat cerebellum, highly enriched in granule cell precursors, to a slow-releasing NO donor (DETA-NONOate) [63]. The morphogenetic factor Sonic hedgehog (Shh) is the main mitogenic determinant for the expansion of the precursor cell population giving rise to mature granule neurons [76]. The essential downstream effector of the Shh signaling pathway in cerebellar development is the oncogenic transcription factor N-Myc [77, 78], which sustains precursor proliferation as demonstrated by the dramatic cerebellar hypoplasia and drop of granule neuron number in transgenic mice with conditional knockout of the gene [79]. In cultures enriched in granule cell precursors, N-Myc responds to pharmacological manipulation of NO levels in a way that exactly parallels the proliferative response of precursors: expression is increased when NOS is inhibited and proliferation is increased while expression is decreased when cells are provided with a continuous exogenous source of NO and proliferation is decreased [26, 63, 75]. Thus, at least in culture condition, NO negatively regulates precursor proliferation through down-regulation of the same target whose up-regulation is essential for the pro-proliferative action mediated by Shh signaling, namely N-Myc. Noticeably, NO supplementation to granule cell precursors in cultures not only decreases the basal level of proliferation but is also able to counteract the increased proliferation caused by exogenous administration of Shh [63], thus strengthening the concept of antagonistic roles in the regulation of granule cell precursor proliferation. It is also interesting to recall the recent demonstration that in a cell line of medulloblastoma, a tumor originated from neoplastic growth of cerebellar granule cell precursors [80, 81] and responding to the cellular cascade Shh/N-Myc for its formation [82], proliferation of tumor cells in culture is slowed down by NO/cGMP signaling [63]. The regulation of N-Myc by Shh in granule cells is not well understood and is complicated by the fact that both direct induction of gene expression and indirect regulation of N-Myc phosphorylation appear to be involved [78]. Furthermore, N-Myc transcriptional activity is also regulated through IGF/PI-3K signaling which inhibits the N-Myc destabilization promoted by GSK3 [78]. The negative regulation of N-Myc mediated by NO functionally opposes the positive regulation of Shh and IGF, is dependent on cGMP/PKG pathway, and has been tentatively associated with a decreased phosphorylation of the protein Retinoblastoma (Rb) which sequestrates the E2F protein whose binding to the N-Myc promoter is essential for transcription of the gene [75]. The diagram of Fig. 1 is a schematic attempt to summarize what is known on Shh, IGF, and NO regulation of N-Myc in cerebellar granule cells.
https://static-content.springer.com/image/art%3A10.1007%2Fs12311-010-0234-1/MediaObjects/12311_2010_234_Fig1_HTML.gif
Fig. 1

Schematic diagram summarizing present knowledge on the interactions among factors regulating neurogenesis of cerebellar granule cells (see text for details). Downward arrow activation, up tack symbol inhibition, dashed lines signaling pathway not yet determined. Gli/Ci transcription factors of the Gli family, GSK glycogen synthase kinase, IGF insulin-like growth factor, IGF-R IGF receptor, PI-3K phosphatidylinositol-3-kinase, Ptch patched, Shh sonic hedgehog, Rb retinoblastoma, Smo smoothened

While, in summary, studies in culture provide convincing evidence for the negative regulation of cerebellar granule cell precursor proliferation by NO, in vivo results have been less conclusive. Transgenic mice lacking one or two NOS isoforms [70, 83, 84] have not been reported to show any gross alteration of the cerebellum in the adult. However, no developmental studies have been so far performed on neurogenesis in the brain of these animals. A study based on continuous inhibition of NOS activity during the postnatal period of granule cell formation and maturation in the rat, similarly failed to reveal any gross alteration in cerebellar anatomy in the adult [27]. However, by studying in neonatal rats' cerebellar development during chronic inhibition of NOS, it was demonstrated that proliferation rate of granule neuron precursors actually responded to pharmacologic NO deprivation, but that this effect was significant only during the first three postnatal days [26]. At the end of this restricted time window of treatment, incorporation of tritiated thymidine and bromodeoxyuridine (BrdU) were increased in the cerebellar external granular layer, and this temporary increase of precursor proliferation could be traced up to adulthood as testified by the increased number of BrdU-positive cells in the internal granular layer of a 60-day-old animal injected with the division marker at the third day of NOS inhibition [26]. Further observations showed that in the same time window of the first three postnatal days, increased precursor proliferation was accompanied by increased expression of N-Myc and of cyclin D1 and that the NO effects were transduced through the cGMP system [26]. In animals in which the pharmacologic treatment was maintained for longer postnatal periods, none of the above effects was seen any more even if NOS activity remained inhibited by more than 90%. While the above study provides proof of principle that manipulation of NO levels during in vivo development alters proliferative neurogenesis of granule neuron precursors in a cGMP- and N-Myc-dependent way, to explain the transitory nature of the effect is not easy. Conceptually, the problem to be faced is analogous to the one posed by the above-remembered absence of adult cerebellar alterations in NOS-knockout mice. Tentatively, it may be hypothesized that the strong reduction of NO (not actually the complete lack of NO production as some NOS activity is still measurable in the brain of knockout mice [8]) is easily overcome by compensatory developmental mechanisms as, for instance, a sustained production of cGMP originated by other cellular mechanisms. That something specific for the developmental stage may affect NO response of postnatal neuronal precursors as compared to those present in neurogenic areas of adult brain, is suggested by the fact that while NOS inhibition increases proliferation among precursors of the subventricular zone, the same response is absent in neonatal animals [85]. At the present stage of investigation, the above-discussed discrepancies remain the most important requiring clarification to completely understand the role of NO on proliferative neurogenesis of neuronal precursors and in particular of precursors of cerebellar granule neurons.

Nitric Oxide in Survival, Differentiation, and Neuroprotection of Cerebellar Granule Neurons

A remarkable piece of research has revealed during the years both neurodegenerative and neuroprotective actions of NO, fully justifying its early denomination of “Janus-faced molecule” [86], the switch between the two opposite actions being determined by a mix among the source and the concentration of NO, the sensitivity of the cells and the whole cellular settings in which the actions take place as documented by several previous reviews [9, 14, 8789]. Regarding cerebellar granule neurons, while several data document obvious detrimental effects of exposure to high NO concentration resulting from exogenous administration or from co-cultured glial cells stimulated with pro-inflammatory signals as well as aggravation by NO of hypoxic/ischemic insult [9096], the present paragraph will focus on physiologic survival promoting actions and on neuroprotection in pathologic conditions, mediated by NO.

As anticipated in a previous paragraph, ability to synthesize NO increases with differentiation in cerebellar granule neurons. In standard culture conditions, NOS activity increases around eightfold between 4 and 8 days in culture and nitrite accumulation in the medium increases at similar rate during the same period [97]. As this time in culture corresponds to the progressive differentiation of granule neurons [29], these data strongly suggest that maturation of NO system is important for granule cell survival and acquisition of mature phenotype. This suggestion was supported by experiments in which cerebellar granule neurons in culture were maintained from the second day in vitro in conditions of pharmacologic inhibition of NOS activity through the broad spectrum inhibitor N-nitro-l-arginine methylester (L-NAME) [27]. While this treatment resulted in no obvious adverse effects during the first 4 days of culture, it determined a significant decrease of the number of cells present in the culture after 8 days in vitro [27]. This observation suggested that NO was necessary to sustain survival of differentiating granule neurons, and further experiments demonstrated that this was actually the case. By exposing cultures of granule neurons after 7 days in vitro to a concentration of L-NAME able to almost completely inhibit NO production, we caused a progressive apoptotic death of cells, reaching 60% after 4 days of pharmacologic treatment [30]. The specificity of the effect, i.e., that it was actually due to NO deprivation and not to some side toxicity of the drug, was demonstrated by the fact that it could be reverted by co-administration of a slow-releasing NO donor [30]. Furthermore, the effect was mediated by NO through the cGMP cascade: bypassing NOS inhibition by providing the cultures with a stable cGMP analog protected cells from apoptosis, while exposing the cultures to an inhibitor of guanylyl cyclase replicated the apoptotic effect of NOS inhibition [30]. Further molecular dissection of the survival pathways activated by NO/cGMP in cerebellar granule neurons led to the identification of two key players: the activation of the Akt/GSK-3 survival kinase system and the activation of the transcription factor CREB inducing expression of anti-apoptotic genes [30, 59].

In addition to the above-described survival promoting action towards granule neurons, NO has been also demonstrated to be neuroprotective in experiments in which the same neurons are challenged with neurotoxic insults. Best-documented neuroprotective effects are those against ethanol neurotoxicity. Exposure of cerebellar granule cells to low ethanol concentration is more toxic to freshly explanted cells than to older cultures, and acquisition of this alcohol resistance depends on the activation of the NO/cGMP/PKG pathway [98]. Neuroprotection given by NO towards ethanol toxicity was confirmed by the greater vulnerability of cerebellar granule neurons derived from nNOS knockout mice and by the rescue granted through activation of the cGMP pathway downstream to NO [99]. Interestingly, a similar result has also been obtained in vivo in a model of rodent microencephaly caused by daily postnatal administration of increasing concentrations of ethanol [100]. Microencephaly was more pronounced, and in particular, depletion of cerebellar granule neurons worsened in mice knockout for nNOS than in wild-type animals of the same age and subjected to the same treatment [100]. Evidence has been reached that the NO-promoted neuroprotection from ethanol in cerebellar granule cells is elicited by well-established pro-survival mechanisms. This is the case for NMDA-mediated and for growth factor-mediated neuroprotection which both act through activation of nNOS [101, 102]. Furthermore, increasing cAMP levels caused by the adenylyl cyclase activator, forskolin, protected granule neurons from ethanol toxicity by eliciting a CREB-dependent increase of nNOS expression [103]. Other models of granule neuron-induced death have been less explored compared to the model of ethanol toxicity. It is, however, interesting that in a model of glutamate-induced excitotoxicity, nitrosothiols GSNO and SNAP were able to protect granule neurons by decreasing the excess entry of calcium through the NMDA receptor suggesting that they modified, possibly through mechanisms of transnitrosation, the calcium permeability of the channel [104]. Furthermore, in similar models of excitotoxicity, as well as in the case of neurotoxic insults of different type, cerebellar granule neurons were protected by carbon monoxide through a pre-conditioning mechanism, i.e. by inducing cellular protective mechanisms after pre-exposure to low concentrations of carbon monoxide itself using NO/cGMP system as downstream mediator [105]. Altogether, the data briefly summarized in this paragraph leave little doubt that in non-pathologic conditions, physiologic amounts of endogenously produced NO are important for granule neuron survival and differentiation and that increased regulation of endogenous NO production or exogenous administration of sub-toxic doses of it may be neuroprotective.

Nitric Oxide and Synaptic Plasticity in Cerebellar Circuits Involving Granule Neurons

As mentioned in the “Introduction”, the diffusible nature of NO and the physiologic link with NMDA receptor activity in cerebellar circuits [5] contributed to his early identification as an appealing candidate for mechanisms of synaptic plasticity. Since then, evidence for the NO/cGMP involvement in plastic changes of synaptic strength, and thus in processes of learning and remembering, has accumulated to a probably unexpected extent [106, 107].

An important form of synaptic plasticity centered on cerebellar granule neurons and involving NO is long-term depression (LTD) occurring in Purkinje neurons when parallel fiber activity is dampened by coincident strong excitatory input of climbing fibers [108, 109]. This mechanism is viewed as a motor learning behavior, whereby climbing fibers signal errors in motor performance by selectively weaken inappropriate motor signals conveyed by granule neurons through parallel fibers [108, 109]. Involvement of NO in LTD, and in particular its cellular source and its targets, have been debated for several years [110116]. A currently widely accepted model is that NO produced by parallel fibers, with possible contribution of molecular layer nNOS-positive neurons, diffuses into Purkinje neurons where it stimulates guanylyl cyclase and activates PKG, contributing to hyperphosphorylation of AMPA receptors which results in their de-clustering and endocytotic recycling, thus lowering excitatory response to glutamate [107, 117119]. Evidence has been produced that induction of LTD at parallel fiber-Purkinje cells synapses requires coincidence of NO production and entry into Purkinje cells with Ca2+ signal elicited in the same neurons by the strong activation derived from climbing fiber input [112, 116, 120, 121]. While LTD seems not to be essential for basal motor learning, it may be involved in specific forms of motor learning such as adaptation of vestibulo-ocular reflex [107]. A major conceptual problem in understanding the specificity of the role of NO in LTD is related to the highly diffusible nature of the molecule. How can specificity be maintained when local production of NO leads to excessive accumulation of it with possible induction of LTD in neighboring synapses? Based on computer simulation, a model has been proposed, whereby NO produced by a limited number of parallel fibers ensures specificity by modifying synaptic strength to a level required for context-dependent selection of internal models of activity, while firing of a fraction of them in inappropriate situations results in level of NO not sufficient for long-term modification of synaptic strength [122]. This simulation essentially supports previous hypothesis based on “volumic” LTD learning rule of NO diffusion in microzones of cerebellar cortex [123].

In addition to the fundamental mechanism of synaptic plasticity subserving LTD-related motor learning, NO is involved in at least one, and possibly two, types of long-term potentiation (LTP) at synapses formed by granule neuron parallel fibers at dendrites of Purkinje neurons. The first type of LTP acts at the same parallel fiber-Purkinje neuron synapses implicated in LTD, is postsynaptic in location, requires a relatively low stimulus frequency for its induction (1 Hz LTP), is coupled with low postsynaptic Ca2+ levels, and has been considered a candidate for reversing LTD, thus ensuring bidirectional plasticity to the parallel fiber-Purkinje neuron transmission [124126]. Another form of LTP, actually the first one reported at parallel fiber-Purkinje neuron synapses, is presynaptic depending on cAMP/PKA signaling [127129], while its possible dependence on NMDA receptor/NO was controversial [130]. This form of LTP requires high-frequency stimulation (4 Hz LTP), is accompanied by presynaptic Ca2+ elevation and increased neurotransmitter release, and a recent report suggests that it is also dependent on activation of NMDA receptor and production of NO [131]. The presence of bidirectional synaptic plasticity, LTD, and LTP, at the same synaptic contacts has suggested that alternation of the two ways to down- or up-regulate synaptic activity could be important to reset the function of the synapses. Supporting this idea, it has been demonstrated that when LTD is saturated by repeated induction, it can be reversed by a single round of LTP or by NO, allowing fresh LTD to be induced again and, conversely, when LTP is saturated, it can be re-induced by LTD [125]. This study allowed to identify postsynaptic LTP mediated by NO as the main mechanism to reverse LTD. Thus, while glutamate is the “classical” transmitter at the parallel fiber-Purkinje neuron synapses, NO produced presynaptically is the diffusible messenger necessary and sufficient to induce LTD, when it is temporally coincident with postsynaptic Ca2+ elevation or LTP when Ca2+ is low in the Purkinje neuron. These mechanisms put NO produced by granule neurons in the position to play a central role in cerebellar learning related to motor control.

It should be also mentioned that the above-summarized mechanisms of plasticity at parallel fiber-Purkinje neuron synapses do not account for the whole spectrum of NO-mediated plastic responses of granule neurons. At the mossy fiber-granule neuron synapses, indeed, a form of LTP can be induced by high-frequency mossy fiber stimulation [132134]. It has been demonstrated that NO is required for induction of LTP at the mossy fiber-granule cell synapses based on the following evidences: (1) during high-frequency stimulation, NO reaches concentrations in the nanomolar range able to fully activate guanylyl cyclase; (2) LTP is blocked by NOS or guanylyl cyclase inhibition; (3) NO donor is able to induce LTP and to maintain it also in the absence of mossy fiber stimulation or in condition of pharmacologic NMDA receptor blockade [135].

Nitric Oxide in Granule Neuron–Glia Crosstalk

Neurons are by no way the only NO-producing cells in the brain. Glial cells, in particular microglia, mostly possess the inducible form of NOS (iNOS), usually present in macrophagic cells, which brings to lasting production of NO upon glial activation with deleterious consequences for neuron health [11, 60, 136, 137]. Due to the fact that inflammatory conditions are characteristic of most acute and chronic neurodegenerative diseases and to the peculiarity of the role played by NO in the inflammatory response by glial cells, many researches have been focused on damage-exacerbating effects of excessive NO production by glial cells. This leaves so far almost unexplored the field of possible functional relationships between neuron- and glia-derived NO [11, 138, 139] as well as the one between NO produced by brain parenchyma and NO produced by the endothelial form of NOS present in endothelial cells of brain vasculature [11].

Regarding cerebellar granule neurons, several studies have been based on co-culture of them with mixed glial or pure microglial cells. In co-cultures, immunostimulated microglia induced death of cerebellar granule neurons, and neurotoxicity was attenuated by inhibition of NOS activity or by NO scavengers, implying a fundamental role of excessive NO production by microglial iNOS in the death response [90, 140]. This could be due to direct neurotoxic effects of NO as well as to induction of autocrine excitotoxicity, in agreement with the fact that NO donors at high concentrations elicit an NMDA dependent neurotoxicity in mature granule neurons in culture [91]. Accordingly, NMDA receptor-mediated toxicity towards cerebellar granule neurons was potentiated by co-culture with immunostimulated microglia or by co-exposure to an NO donor [141]. Further confirmation of the aggravating role of excessive NO production from activated glia comes from studies demonstrating exacerbation of damage due to excitotoxic-like conditions, i.e., glucose deprivation and hypoxia [94, 137, 142]. Altogether, these data suggest that inflammatory conditions lead glial cells, and in particular microglia, to assume a neurodestructive phenotype and that induction of iNOS and overproduction of NO play a central role in the neurodegenerative mechanism. While involvement of excessive NO production from activated glia as a neurodamaging factor may be directly demonstrated in culture by measuring NO endproduct accumulation in the medium and by the use of appropriate inhibitors and scavengers, a similar demonstration in vivo may be more complicated. This increased complication is linked to the occurrence in vivo of several natural scavengers, such as hemoglobin of circulating red blood cells and other biological molecules able to react with NO contributing to lower its concentration. The intriguing relationships among neuroinflammation, NO production and neuronal damage open interesting perspectives for research aimed to devise ways to control inflammatory state of microglia in neurodegenerative diseases or, even more exciting, to drive neuroinflammation towards neuroprotection [143147].

It is, however, clear that interactions between glia and neurons are two-way and this is in particular is true regarding microglia and neurons [148]. Some of these reciprocal interactions have been demonstrated between microglia and cerebellar granule neurons and in some cases NO has been shown to play a role. Through the use of conditioned media from granule cell cultures, evidence has been provided that differentiating neurons release in the medium substances able to control the inflammatory state of microglia and to induce apoptotic elimination of immunostimulated microglia [149]. That NO plays a critical role in the control of inflammatory activation of microglia by cerebellar granule neurons was shown by a study in which co-cultures of granule cells-microglia or exposure of microglia to a medium previously conditioned by mature granule neurons induced LPS-stimulated microglia to greatly increase the expression of iNOS and the production of NO [150]. The true inflammatory nature of the increased response of microglia, exposed to LPS and concomitantly to mature cerebellar granule neurons or their conditioned medium, was confirmed by the fact that also another typical pro-inflammatory marker, interleukin-1β, underwent increased production and release in the medium [150]. Prolonged (72 h) exposure to neuron-conditioned medium in the presence of LPS induced microglia apoptosis [150]. This could be interpreted as a safety mechanism whose nature should be further studied in vivo and in which the granule cell-stimulated iNOS induction and NO production by microglia appeared to be central players. The above-described research is starting to clarify the complexity of the cerebellar granule neuron–microglia interactions involving reciprocal regulation of NO production by the respective forms of NOS present in the two cellular types. From our recent observations, indeed, it appears that in co-culture conditions, the expression of nNOS in cerebellar granule neurons on which microglia has been freshly plated is strongly reduced during the first hour of co-culture, likely due to the activation of a specific proteolytic mechanism and remains significantly depressed up to 24 h of co-culture [Polazzi et al., in preparation]. This intriguing result further highlights the interest of studying more in deep the functional meaning of reciprocal control of NOS expression and NO production exerted by microglia over neurons and by neurons over immunostimulated microglia. As pointed out before, we still know very little regarding non-pathologic NO-related interactions between neurons and glial cells. These first data on granule neurons and microglia suggest that this model could be appropriate to start investigating how reciprocal control of NO production may impact on functional relationships between neurons and microglia.

Concluding Remarks and Future Perspectives

The present short review of available data points to the great impact of NO-related cellular mechanisms for almost every aspect of life cycle of cerebellar granule neurons as well as for their essential functions in cerebellar circuitry. It is remarkable to find that NO and its associated signaling is important for these cells from early stages of development, when it regulates the proliferation of their precursors, to more advanced stages of development, when NO promotes their survival and differentiation, and finally to the mature situation, when NO is life-long essential for plasticity and tuning of granule neuron-centered cerebellar circuitry subserving motor learning. While remarkable, this situation may not actually come to a surprise for people familiar with NO research. More than 20 years of research on this molecule have, indeed, demonstrated its multifarious nature and its involvement in many, if not all, main organism functions (circulation, respiration, digestion, reproduction, immunity, brain function etc.) as well as in several important pathologies (neurodegenerative, inflammatory and cardiovascular diseases, cytotoxicity, tumors etc.). To restrict ourselves to the nervous system, it is important to stress that cerebellar granule neurons are a privileged neuronal population in which to study NO in its function and dysfunction. These neurons constitute the largest homogeneous neuronal population of the whole brain and are endowed with an exceptionally high capacity to produce NO and to respond to NO signaling for their fundamental functions in every stage of their life. There is, therefore, little doubt that granule neurons could represent a model of election for studies on the various functions of NO in brain physiology and of its involvement in brain pathology. Among the various issues considered in the present review, some will likely constitute the main object of future research. In my personal opinion, these will be: (1) a better understanding of the exact role of NO in granule cell neurogenesis and of the interactions with other regulators of neurogenesis; (2) a remarkable expansion of studies aimed at the comprehension of the mechanisms through which NO regulates gene expression acting both at the level of transcription factors and of epigenetic regulation; (3) involvement of NO in tumorigenesis of medulloblastoma and possible identification of NO/cGMP system as a potential therapeutic target for these tumors. Thus, while the past 20 years of research have marked an impressive improvement of our understanding of NO functions in cerebellar granule neurons, future perspectives of research on this issue promise to be even more productive and exciting.

Acknowledgement

Work done in the author's laboratory reviewed in the present paper, has been funded by grants of the Italian Ministry for Universities and Research (PRIN and FIRB schemes) and by research funds of the University of Bologna.

Conflict of Interest

The author declares that there are no conflicts of interest related to the present paper.

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© Springer Science+Business Media, LLC 2010