Purinergic neuron-glia interactions in sensory systems - Springer Link

Pflugers Arch - Eur J Physiol (2014) 466:1859–1872 DOI 10.1007/s00424-014-1510-6

INVITED REVIEW

Purinergic neuron-glia interactions in sensory systems Christian Lohr & Antje Grosche & Andreas Reichenbach & Daniela Hirnet

Received: 19 March 2014 / Revised: 26 March 2014 / Accepted: 26 March 2014 / Published online: 6 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The purine adenosine 5′-triphosphate (ATP) and its breakdown products, ADP and adenosine, act as intercellular messenger molecules throughout the nervous system. While ATP contributes to fast synaptic transmission via activation of ionotropic P2X receptors as well as neuromodulation via metabotropic P2Y receptors, ADP and adenosine only stimulate P2Y and P1 receptors, respectively, thereby adjusting neuronal performance. Often glial cells are recipient as well as source for extracellular ATP. Hence, purinergic neuron-glia signalling contributes bidirectionally to information processing in the nervous system, including sensory organs and brain areas computing sensory information. In this review, we summarize recent data of purinergic neuron-glia communication in two sensory systems, the visual and the olfactory systems. In both retina and olfactory bulb, ATP is released by neurons and evokes Ca2+ transients in glial cells, viz. Müller cells, astrocytes and olfactory ensheathing cells. Glial Ca2+ signalling, in turn, affects homeostasis of the nervous tissue such as volume regulation and control of blood flow. In addition, ‘gliotransmitter’ release upon Ca2+ signalling—evoked by purinoceptor activation—modulates neuronal activity, thus contributing to the processing of sensory information.

C. Lohr (*) : D. Hirnet Division of Neurophysiology, Biocenter Grindel, University of Hamburg, 20146 Hamburg, Germany e-mail: [email protected] A. Grosche (*) Institute of Human Genetics, Faculty of Medicine, University of Regensburg, 93053 Regensburg, Germany e-mail: [email protected] A. Reichenbach Paul Flechsig Institute of Brain Research, Faculty of Medicine, University of Leipzig, 04109 Leipzig, Germany

Keywords ATP . Adenosine . Purinoceptors . Retina . Olfactory bulb . Müller cells . Ensheathing cells . Astrocytes

Introduction Adenosine 5′-triphosphate (ATP) not only is a ubiquitous cellular energy carrier, it also serves as a signalling molecule between cells. ATP is released from many types of cells upon chemical or electrical stimulation, including cells of the nervous system [9]. ATP release pathways include exocytosis, transporters and membrane pores such as gap junction hemichannels and volumeregulated anion channels. Once released, ATP is detected by purinoceptors of the P2 family which are classified into ionotropic P2X receptors (P2X1–P2X7) and metabotropic P2Y receptors (P2Y1, 2, 4, 6, 11, 12, 13, 14 in mammals) [10]. The purinergic system is more versatile than most other neurotransmitter systems, since extracellular degradation of ATP is far from producing immediate inactivation of the system. The first breakdown product, ADP, is a potent agonist at some P2Y receptors, while the end product of ATP dephosphorylation, adenosine, activates another family of purinoceptors, the metabotropic P1 receptors (comprising A1, A2A, A2B and A3). Different extracellular enzymes such as ectonucleotidases and tissue-nonspecific alkaline phosphatase catalyze certain steps of ATP degradation [149], and the combination of tissue-specific purinoceptors and enzymes makes purinergic transmission a signalling system with many facets. Purinergic neurotransmission has been extensively studied in the peripheral nervous system, e.g. between parasympathic nerves and smooth muscle cells [8], as well as the central nervous system [61, 64]. The first hint for purinergic transmission was found in sensory nerves decades ago [45]; since then, it turned out that purinergic neurotransmission is a key player in information processing in most if not all sensory systems. ATP modulates neurotransmission in the cochlea, mediates pain perception and is even the principal transmitter

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between taste cells and nerve endings perceiving taste information and propagating it into the brain via the gustatory nerve [30, 46, 114, 136]. Glial cells are equipped with a plethora of purinoceptors and are often targets of purines released by neurons in sensory organs (such as the retina and the olfactory epithelium) as well as in brain areas processing sensory information, but can also release ATP by themselves, thereby modulating sensory information processing [47, 91, 118, 124, 144]. In this review, we will focus on the mechanisms and functions of purinergic neuron-glia communication in two sensory systems, the visual system and the olfactory system.

Purinergic signalling in the retina Retinal structure The retina comprises the light-sensitive photoreceptors which translate visual stimuli into neurochemical signals. Subsequently, this information is communicated to the second cell type in the phototransduction pathway, the retinal bipolar cells. The latter pass the incoming signals to the third neuron in this signalling cascade—the retinal ganglion cells—which finally forward the information to higher centres in the brain. The dominant transmitter promoting this ‘vertical’, direct flow of information is glutamate. Additionally, a first intraretinal integration of visual information is mediated by the activity of amacrine and horizontal cells modulating synaptic transmission in the inner and outer plexiform layer, respectively. Transmitters involved in these ‘transversal’, integrative networks are γ-aminobutyric acid (GABA), acetylcholine, dopamine, glycine and, most likely, also ATP. Interestingly, glial cells might also directly contribute to retinal information processing. One main ‘avenue’ of neuron-glial communication in this context turned out to be organized via purinergic signalling mechanisms [83, 87, 100]. Both retinal neurons and glial cells are endowed with various purinergic receptors enabling them to respond to changing levels of extracellular purines including ATP, ADP and adenosine (Figs. 1 and 2). With their stem processes spanning the whole thickness of the neural retina and myriads of fine processes emanating from them, Müller cells, the principal macroglia cells of the retina, are in intimate contact with every neuronal structure (e.g. with the synapses in the plexiform layers), with blood vessels, the vitreous and the subretinal space (Fig. 2). Consequently, Müller cells are perfectly equipped to mediate neurovascular coupling, but also to directly affect neuronal activity and synaptic transmission by the release of ‘gliotransmitter’ such as glutamate, D-serine, GABA and ATP [97]. In the following sections, we will delineate how purinergic signalling is involved in glia-neuron-interactions in the retina.

Pflugers Arch - Eur J Physiol (2014) 466:1859–1872

Expression of purinergic receptors on retinal neurons All retinal neurons express a variety of purinergic receptors, belonging to the families of P1 and P2 receptors, as comprehensively reviewed by Housley et al. [47]. Due to the complex, partially species-specific expression pattern of receptor (sub-)types, the functional relevance of purinergic signalling is still unsatisfactorily understood. Activation of ionotropic P2X receptors by ATP appears to contribute to fast modulation of neuronal information processing [94]. Interestingly, some P2X receptors, such as the P2X2 receptor, are enriched in specific retinal circuits. Activation of P2X2 on OFFamacrine cells increases inhibitory GABAergic postsynaptic currents, thereby decreasing the responses of OFF-ganglion cells and promoting the responses of ON-ganglion cells [56, 57]. Moreover, there is evidence that P2X2 receptors are preferentially involved in mediating the cone responses, while P2X7 receptors seem to shape both the rod and cone response patterns [94–96, 125]. Recently, P2X7 has been shown to be expressed primarily on neurons and astrocytes of the inner retina, but not on Müller glia cells [125]. In P2X7-deficient animals, the response of rod photoreceptors (a-wave) was found to be unaltered, whereas the rod-driven postphotoreceptor response (b-wave) was accelerated in amplitude and speed. The same was observed in the cone signalling pathway which implies that the depolarization of rod and cone bipolar cells is enhanced upon light stimulation in mice deficient for P2X7 [125]. Accordingly, the authors speculate about an excitatory input of P2X7 on photoreceptor terminals or on inhibitory neurons in the inner nuclear layer, modulating the response patterns of second-order neurons in the cone and rod signalling pathways. Regarding the functional expression of metabotropic purinergic receptors, there are results from various immunolabelling and in situ hybridisation studies suggesting an expression of P2Y1 receptors (highly sensitive to ATP and ADP) on GABAergic, cholinergic and dopaminergic amacrine cells and in retinal ganglion cells [19, 32, 132]. The dominant P2Y receptor on rod bipolar cells, which is dominantly localized to their synaptic endings (but also found on their somata and dendritic trees), is the P2Y4 receptor (responsive to UTP) [133, 141]. In addition to the P2X7 receptor [88, 125], photoreceptors express P2Y6 which is predominantly activated by UDP [148]. An expression of P2Y6 has also been reported for horizontal, amacrine and ganglion cells, but not for bipolar or Müller glia cells [132, 148]. However, up to now, very little is known about the functional relevance of these receptors in the various retinal cell types. Considering studies on the impact of purinergic modulation on the conductivities of ion channels such as voltage-gated calcium and potassium channels as well as ligand-gated NMDA channels [1, 5, 6, 28, 29, 34, 76, 135], it is very


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Fig. 1 Artist’s view of how purinergic signalling is involved in glial function. Volume regulatory signalling cascade of Müller glia as confirmed by pharmacological measurements in vital retinal slices [110, 143]. Arrows pointing to the retinal neurons indicate putative paracrine actions via gliotransmitters (green arrows). Vice versa, overspill of neurotransmitters (blue arrows) from synaptic sites might stimulate the glial signalling cascade, by acting on the metabotropic receptors of the glial cells. See text for details. 5′NT 5′ectonucleotidase, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, PRS photoreceptor segments

likely that their function in the retina involves the finetuning of the activity in distinct retinal circuits [148]. Most of the retinal cell types also express adenosineresponsive P1 receptors, though the functional relevance of their presence in the respective cell type(s) is largely undefined [47]. Newman found conclusive evidence for the expression for Gi-coupled A1 receptors in retinal ganglion cells, where their activation reduces light-evoked spike activity in the majority of the cells (Fig. 2) [84]. Gs-coupled A2A receptors are also expressed on presynaptic structures of photoreceptors modulating light and dark adaptation by enhancing the phosphorylation of connexins and, thus, the highly variable cone-rod photoreceptor coupling via gap junctions (Fig. 2) [72]. Purinergic receptor expression and function in differentiating and mature Müller cells During retinal development, Müller cells are the last to differentiate out of the late radial glia-like progenitor cells; accordingly, their proliferation rate is still high during the first

postnatal week in laboratory rodents [12, 145]. It is not before the end of the second postnatal week—coinciding with the time point of eye opening—that the cells start to establish their well-characterized physiological features such as a pronounced potassium conductance and a tight regulation of their cellular volume [139]. Purinergic signalling is involved in a plethora of processes in the retina including the regulation of cell proliferation [79, 107, 144], cell differentiation and modulation of neuronal information processing [56, 83, 87]. In rat Müller cells, only two P2 receptor types could be unequivocally identified, both belonging to the subgroup of G-proteincoupled P2Y receptors. First, functional and immunohistochemical data identified the P2Y1 receptor as the principal P2Y receptor present in Müller cells, being of outstanding importance for Müller cell functions such as volume regulation [141]. Its expression in Müller cells could be detected as early as at postnatal day 5 (P5) and in all successive developmental stages right into adulthood [141]. Activation of P2Y1 accounts for ATPinduced calcium increases in Müller cells. The Ca2+ response was blocked by co-administration of MRS2179, a specific

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Fig. 2 Artist’s view of how purinergic signalling is potentially involved in neuron-glia crosstalk. Examples for putative modulatory feedback mechanisms via gliotransmitter release (green arrows) stimulated by intense neuronal activity (neuronal glutamate or ATP release, blue arrows). Upper inset, microdomain 1 (MD1) of the Müller cell is a process enwrapping the synaptic contact between a bipolar cell and a ganglion cell in the inner plexiform layer (IPL). According to what was described in Newman [83, 86], neurotransmitters such as ATP or glutamate stimulate glial P2Y1 and mGluR receptors, thereby initiating the release of adenosine (AD, either directly via nucleoside transporters (NT) or by glial ATP release and its extracellular conversion by ectonucleotidases (eNTs) to adenosine). Adenosine then acts back, for example by binding on A1 receptors of the postsynaptic element (i.e. of a ganglion cell). A1 receptor stimulation on retinal ganglion cells was shown to cause a reduced firing rate of these output neurons, by stimulating barium-sensitive potassium channels (thus eliciting a more hyperpolarized membrane potential of the ganglion cells) [83]. Another process of the Müller cell (MD2) ensheathes an electric synapse between an amacrine and a bipolar cell. Little is known about the effects of purinergic signalling at these interaction sites. Still, amacrine cells are likely a source of ATP; they also have been shown to express P2Y1

receptors. Thus, a neuron-glia crosstalk might take place at this level as well. Note that events in each of the Müller cell microdomains can occur independently from one another, modulating the neuronal information processing with high specificity also in terms of spatial resolution. In contrast, during neuronal hyperexcitation, the principle of functional glial microdomains might be overrun, and a more general, unspecific, glial response might result. Lower inset, hypothetical contribution of Müller glia to the modulation of photoreceptor coupling in the course of light– dark adaptive processes. Li et al. [72] documented that activation of A2A receptors on photoreceptor terminals leads to an enhanced phosphorylation of gap junction proteins by protein kinase A (PKA) under dim light conditions; this increases the retinal sensitivity for incident light. As glutamate (blue arrows) is constantly released at the photoreceptor synapse in the dark, the surrounding Müller cell processes might also receive a persistent input by stimulation of their metabotropic (group II) glutamate receptors. This, in turn, is likely to result in a tonic adenosine release (green arrow) via nucleoside transporters (NT) from the cells, facilitating the photoreceptor coupling by additional activation of A2A receptors. BV blood vessel, GCL ganglion cell layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer

P2Y1 receptor antagonist [120, 141], while other P2Y antagonists such as reactive blue 2 (primarily blocks P2Y4) or MRS2578 (a specific antagonist of P2Y6) were ineffective. Moreover, among other purines such as UTP or UDP, which preferentially bind to P2Y4 and either P2Y2 or P2Y6, only

UTP elicited a small Ca2+ response in adult Müller cells. In immature cells, neither UTP nor UDP application resulted in a specific Ca2+ increase. Interestingly, the calcium response pattern of Müller cells evolves during development. While ATP induces a transcellular calcium wave in immature Müller


cells, spreading from the endfoot—passing the inner stem process and the soma—into the outer stem process, in mature cells, the response remains restricted to the endfoot region [71, 141]. In consequence, the percentage of responding Müller cell somata dramatically declines during the first three postnatal weeks from a maximum of 60 % at P10 to 16 % at P25, finally reaching only 2 % in the adult. Immunohistochemical data might provide one explanation for these data; these indicate that the local density of P2Y1 receptors is higher in the endfeet than in the somata, a difference that appears to increase with postnatal age. However, other mechanisms— such as an enhanced expression of calcium-binding proteins as a result of Müller cell differentiation—might contribute to the altered Ca2+ response, as well. In addition to the P2Y1 receptor, Müller cells express a second P2Y receptor, P2Y4. Although being coupled to Gq/11, like P2Y1, it only marginally contributes to Ca2+ responses in (differentiated) Müller cells and is not involved in their volume regulation at all. In contrast to P2Y1 and in line with functional data, apparent expression of P2Y4 is not demonstrable before the end of the third week (P20). It is mainly localized to Müller cell endfeet, but occurs also in the synaptic boutons of rod bipolar cells. At P20, Müller cells already have established their principal adult-like physiological and morphological features [139]. Moreover, all retinal neurons are born and the neuronal circuits are set. Of note, the end of the third postnatal week (i.e. the time of increased P2Y4 expression) also marks the onset of maturational processes leading to refinement of neuronal interactions, including the conversion of bistratified ON–OFF responsive retinal ganglion cells to monostratified ON and OFF responsive retinal ganglion cells [27, 117, 137]. Noteworthy, an activation of the PI3K/Akt pathway was shown in P2Y4-positive bipolar cells [141]. Considering that activation of this pathway is involved in mechanisms regulating synaptic stability [49, 78, 119, 131], it is tempting to speculate that P2Y signalling might contribute to neuron-to-glia interactions which promote synaptogenesis in the rat retina. Finally, the functional expression of one P1 receptor, viz. the A1 receptor, has been documented (Fig. 2) [8, 142]. This Gi-coupled receptor for adenosine is of outstanding functional relevance for Müller cells, as its activation constitutes the final step in the complex glutamatergic-purinergic volume regulatory cascade of Müller cells (see below). Sources of purines in the retinal circuitry In principle, neurons as well as glia cells might serve as potential sources of signal-mediating purines. However, an ultimate proof of the native release, and of its source(s), is still pending for most of the candidate molecules. Regarding ATP release, the most likely neuronal structures are GABAergic horizontal cells and photoreceptor synapses in the outer plexiform layer, and GABAergic amacrine cells in the inner

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plexiform layer. GABAergic amacrine cells co-release ATP with their ‘major’ transmitter via calcium-dependent exocytosis [94]. The retinal expression of the recently discovered vesicular transporter of ATP (VNUT), a prerequisite for exocytotic ATP release, has been documented for both inner retinal neurons and photoreceptors [125]. Moreover, Newman concludes from data of antidromic ganglion cell stimulation that retinal ganglion cells are capable to induce intracellular calcium transients in neighbouring Müller glia cells by release of ATP [85]. Unlike ATP, adenosine is not considered to be a conventional transmitter in the retina; rather, it is assumed to act as a neuromodulator [15]. Adenosine exists free in the cytoplasm and is probably shuttled in and out of the cell via equilibrative nucleoside transporters according to the concentration gradients (ENTs) [16, 53, 75, 130]. Alternatively, there exists an indirect ‘adenosine release pathway’, where adenosine is generated by extracellular conversion of ATP via the activity of ectoenzymes such as NTPDase1 or 2, and finally, 5′-ectonucleotidase. Of note, NTPDase2 appears to be the dominant ATP-converting enzyme in the healthy retina. It degrades ATP to ADP but not down to AMP; this step requires the activity of 5′ectonucleotidases which indeed have been demonstrated in the retina [48]. In the diabetic retina, this picture changes remarkably; the NTPDase1 is up-regulated, leading to an accelerated degradation of ATP via ADP to AMP and, consequently, to a faster degradation of ATP to adenosine [140]. Interestingly, these enzymes are primarily located on Müller glia cells, probably indicating the high importance of a tightly regulated purinergic signalling at the neuron-glia interface. Complementing these two well-accepted mechanisms of adenosine release, a recent report provided first conclusive evidence that adenosine might also be set free via neuronal vesicles from parallel fibres in the hippocampus [62]. Whether this mode of adenosine release is also active in retinal neurons remains the subject of future research. Müller cells, as the only macroglia cells in the retina being in intimate contact with neuronal synapses, are capable of releasing several gliotransmitters such as glutamate, ATP and adenosine. Indeed, the release of ATP (presumably via hemichannels, but not via calcium-dependent exocytosis [7, 110]) and adenosine (via ENTs, not via exocytosis [110, 140, 143]) are mandatory steps in the course of the Müller cell volume regulation where both transmitters act in an autocrine manner. Extracellular conversion of ATP to ADP by NTPDase2 is crucial for the signalling cascade to work [48, 140] although there are species differences. Whereas adenosine involved in glial volume regulation appears to be exclusively released via ENTs in rats, concurrent extracellular conversion of ATP and ADP to adenosine via 5′-ectonucleotidase (CD73) is of relevance in mice [140, 143]. Additional to their role in volume regulation, these release mechanisms can be stimulated by various factors acting upstream of the P2Y1 and

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A1 receptor. For example, glutamate—released either from neurons or from the Müller cells themselves—induces ATP efflux from Müller cells, via stimulation of metabotropic glutamate receptors on the cells [140]. To make the story even more complex, a whole library of trophic factors including vascular endothelial growth factor (VEGF), heparin-binding epithelial growth factor (Hb-EGF), osteopontin, erythropoietin, testosterone and neuropeptide Y (NPY) elicit a glial glutamate release and, consecutively, also a release of ATP and adenosine [66, 82, 121, 129, 134, 140]. For all these signalling molecules, a neuroprotective effect has been described in various injury models. These effects might partially be due to their action on Müller cells, e.g. supporting their homeostatic function and inducing the release of gliotransmitters such as adenosine for which beneficial actions on neurons have been described during various neurodegenerative diseases [73, 89, 105, 146]. Implications for purinergic neuron-glia interactions: volume regulation and synaptic modulation Via a process known as spatial potassium buffering, Müller cells clear the extracellular space from surplus potassium ions to allow repetitive neuronal activity [60, 63]. According to the osmotic gradient, water follows potassium into the Müller cells which then counteract volume changes by release of potassium and osmotically obliged water at contact sites to big fluid-filled compartments such as blood vessels and the vitreous body. An efficient volume regulation of Müller is dependent on (i) their high potassium conductance (mediated by Kir2.1 and Kir4.1 potassium channels) and (ii) the activity of a glutamatergic-purinergic signalling cascade [90, 138] (Fig. 1). Assuming that an onset of the glial volume regulation machinery—with the associated gliotransmitter release—is a consequence of intense neuronal activity, it is tempting to speculate that this gliotransmitter release might not only mediate the autocrine maintenance of a normal Müller cell volume, but also represents a direct feedback mechanism from Müller glia to neurons. As Müller cells enwrap neuronal structures (including synapses) in all retinal layers, it appears reasonable to assume that the model of glial microdomains [36, 98] also applies to the retina. This would allow a single Müller cell to modulate the synaptic strength in the outer plexiform layer in one way, while, in parallel, distinct glianeuron interactions of the same cell occur at synaptic contacts in the inner plexiform layer (Fig. 2). In fact, flickering light stimuli (inducing maximal neuronal activity) or antidromic ganglion cell stimulation results in an enhanced frequency of calcium transients in Müller glia, due to an ATP release from neurons of the inner retina [85, 100] (in contrast, no calcium rises were detected in the astrocytes which reside in the nerve fibre layer and, thus, are far from transmitter release sites [84]). These intracellular calcium rises


always originated in the inner Müller glia stem process and spread into the endfeet, but never reached the somata of the cells or the outer plexiform layer [85]. Consequently, the glial feedback—in this case, via adenosine generated from glialderived ATP, acting on neuronal A1 receptors—is likely limited to the inner retina. The spatial restriction of calcium rises in Müller cells is in line with the developmental data presented above, as the ATP-induced response of the cells becomes constrained to their endfeet and inner stem processes during postnatal differentiation [141]. In fact, it has recently been discussed for cortical astrocytes on the basis of an in vivo study [35, 113] that a general (‘whole-cell’) calcium rise upon stimulation in undifferentiated glia becomes limited to calcium responses of glial microdomains in mature glia; thus, this might be a common developmental process in glial cells. An important molecular basis for this might be the redistribution of involved receptors to microdomains, such as described for the mGluR5 receptor in cortical astrocytes [113] or the P2Y1 receptor in Müller glia [120, 141]. One can only speculate about putative functional implications of a link between neuronal activity and gliotransmitter release from Müller glia, in the course of stimulation of their volume regulatory signalling cascade (by neuronal glutamate or ATP). Due to technical limitations, measurements determining the mechanisms of Müller cell volume regulation hitherto can only reveal changes in their somata, i.e. away from synaptic sites. Imaging of morphological alterations of their perisynaptic processes in response to neuronal excitation would help to further elucidate the complex pathways of neuron-glia interactions, including the contribution of purinergic signalling. Yet, adenosine prevents glutamateinduced bipolar cell swelling [127]. As the bipolar cell somata lay in close proximity to Müller glia somata in the inner nuclear layer, this finding might explain how glial adenosine can directly support or maintain neuronal functions under conditions of glutamate-induced neurotoxicity. Another potential interaction might occur between Müller glia and photoreceptor synapses. There, the A2A receptor was suggested to maintain the coupled state of photoreceptors via gap junctions, potentially to optimize the sensitivity of the rod system under dim light conditions [72]. In response to a constant glutamate release from photoreceptors [38], Müller cells might tonically release adenosine towards active photoreceptor synapses, thereby stabilizing photoreceptor coupling and, thus, retinal sensitivity at dim light.

Purinergic neuron-glia signalling in olfaction Purinergic effects on neurogenesis in the olfactory epithelium Olfactory sensory neurons are located in the olfactory epithelium in the nasal cavity and project their axons (olfactory


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sensory axons) through the cranium into the olfactory bulb (Fig. 3a). Sensory neurons in the olfactory epithelium are burdened with bacteria, viruses and dust from the respiratory air and, hence, undergo apoptosis after a short life time of a few weeks. They are replaced by new neurons which are built from stem cells residing in the nasal epithelium and then have to grow their axons from the epithelium into the olfactory bulb. This regenerative potential is exceptional, since ingrowth of axons from the peripheral into the central nervous system is restrained in other parts of the mature mammalian nervous system. Activation of purinoceptors increases the proliferation rate in the olfactory epithelium and thereby supports regeneration [50]. ATP is released via exocytosis in the olfactory epithelium and activates both P2X and P2Y receptors [42, 43]. In addition to sensory neurons, supporting glial cells in the olfactory epithelium (sustentacular cells) express P2Y receptors and respond to ligands of P2Y2 receptors [20, 40, 43, 44]. Sustentacular cells contain NPY, and purinergic

calcium signalling in the olfactory epithelium has been shown to enhance gap-junctional coupling between sustentacular cells and to stimulate the release of NPY [39, 58, 126]. Since NPY supports neogenesis of olfactory sensory neurons, P2Y2mediated release of NPY from sustentacular cells may account for the proliferative effect of ATP [51, 58].

Fig. 3 Glial cells in the olfactory bulb: a longitudinal section of a mouse head, highlighting the olfactory epithelium (OE) in the nasal cavity and the olfactory bulb (OB). b Periglomerular astrocytes (pAC) extend processes into the neuropil of a single glomerulus, comprising synapses between axons of olfactory sensory neurons (OSN), mitral cells (MC), tufted cells (TC) and periglomerular interneurons (IN). Bundles of OSN axons are enwrapped by olfactory ensheathing cells (OEC). In the external plexiform layer (EPL), synapses between mitral/tufted cells and granule cells (GC) are accompanied by processes of astrocytes (AC). NL nerve layer, GL glomerular layer, MCL mitral cell layer, IPL internal

plexiform layer, GCL granule cell layer. c Axon-glia interactions in the olfactory bulb. Olfactory sensory neurons release glutamate and ATP by exocytosis along the axons in the nerve layer and at synaptic terminals in glomeruli. In OECs in the outer nerve layer, glutamate and ATP bind to mGluR1 and P2Y1 receptors, respectively, leading to calcium release from the endoplasmic reticulum (ER). OECs in the inner nerve layer are not responsive to ATP and glutamate. In olfactory bulb astrocytes, glutamate stimulates mGluR5. ATP stimulates P2Y1 receptors and is then degraded to ADP and, finally, adenosine. Adenosine evokes an increase in calcium by activation of A2A receptors

Cellular architecture of the olfactory bulb The mammalian olfactory bulb is a highly structured area of the forebrain with several morphologically and functionally distinct layers (Fig. 3b). Glial cells are specialized to cope with the different demands of each layer concerning both development and physiology. Olfactory ensheathing cells (OECs), a unique type of glial cells, accompany axons of olfactory sensory neurons from the sensory epithelium to the olfactory bulb, thereby ensheathing bundles of several hundreds of axons [24]. In the olfactory bulb, OECs populate the most

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superficial layer, the nerve layer. In addition, the nerve layer comprises olfactory sensory axons, but is devoid of neuronal cell bodies, dendrites and synapses [3]. OECs in the nerve layer can be divided into two subpopulations, based on their position in the nerve layer and the expression of marker proteins. For instance, OECs in the outer part of the nerve layer express the low-affinity nerve growth factor receptor P75 [3, 116]. They promote axon growth of new-born sensory neurons in the olfactory epithelium and, thereby, enable these axons to pass the border between the peripheral and the central nervous system, a process that is suppressed in other parts of the nervous system [25, 112]. OECs in the inner part of the nerve layer, which lack the P75 receptor, enhance the motility of growth cones and support the guidance of axons into the specific glomeruli of their destination [112]. In the glomerular layer, receptor axons synapse onto dendrites of local interneurons as well as of mitral and tufted cells [4, 18, 26], which are often addressed as a functionally homogenous cell population and are therefore named MT cells. The neuropil within the glomeruli is pervaded by a dense meshwork of processes of periglomerular astrocytes [17, 122]. Astrocytes of the same glomerulus are coupled by gap junctions, while astrocytes sending processes to different glomeruli are not, highlighting that each glomerulus is a functional unit [102]. Cell bodies of mitral cells build the mitral cell layer and give rise to several lateral dendrites that invade the external plexiform layer. These are connected via reciprocal synapses to granule cells, the cell bodies of which are densely packed in the central layer of the olfactory bulb, the granule cell layer. The external plexiform layer and granule cell layer comprise astrocytes that morphologically resemble astrocytes in other parts of the forebrain. Purinoceptor expression in the olfactory bulb Purinoceptor expression has been demonstrated throughout the entire olfactory bulb by means of in situ hybridisation, antibody labelling and the use of radiolabelled receptor ligands. mRNA of all four adenosine receptor subtypes was detected in olfactory bulb tissue by Northern blot [21, 77]. The presence of A1 and A2A receptors was confirmed by in situ hybridisation, antibody staining and radioligand binding assays [52, 55, 101]. The significance of adenosine in the olfactory bulb is emphasized by high activities of enzymes involved in adenosine production, such as 5′-nucleotidase and tissue-nonspecific alkaline phosphatase, as well as of enzymes mediating adenosine breakdown, such as adenosine deaminase [13, 33, 68, 103, 104]. In contrast to the well-described expression of adenosine receptors, information about the expression of P2Y receptors in the olfactory bulb is hardly found in the literature. Simon and co-workers used a radiolabelled P2Y1 ligand to investigate the distribution of P2Y1 receptors in the olfactory bulb [108]. P2Y1like staining was found throughout all layers of the olfactory


bulb, with the most intense staining in the glomerular layer. In addition to P2Y1 receptors, P2X receptors have been shown to be expressed in the olfactory bulb. In situ hybridisation revealed expression of P2X4, P2X5 and P2X6 in the principal neurons of the olfactory bulb, mitral cells and tufted cells [14, 37, 128]. These neurons—but not glial cells—are also immunopositive for P2X2, P2X4 and P2X5 [37, 59, 70]. Interestingly, despite the strong expression of P2X receptors in mitral cells, P2Xmediated currents could not be measured so far, while rapid photolytic application of ATP from ‘caged’ ATP evokes P2Y1mediated inward currents in mitral cells [31]. Purinergic signalling in astrocytes Notwithstanding the fact that purinoceptors have been detected histologically in the olfactory bulb for a long time, functional expression of purinoceptors has been established only recently. Two types of purinoceptors are linked to intracellular Ca2+ signalling in periglomerular astrocytes, the P2Y1 receptor and the A 2A receptor (Fig. 3c) [22]. In addition, periglomerular astrocytes express metabotropic glutamate receptors type 5 (mGluR5) and, hence, are able to respond with Ca2+ rises to both ATP and glutamate that are released from the terminals of receptor axons in the glomeruli [93, 123]. ATP does not only activate astrocytic P2Y1 receptors, but is also degraded by extracellular enzymes, yielding adenosine. The activity of these enzymes is particularly high in the olfactory bulb, allowing for a rapid and complete conversion of ATP to adenosine [68]. Since most glomerular astrocytes in the olfactory bulb express both P2Y1 and A2A receptors, they respond to ATP as well as to adenosine [22]. The A2A receptor-dependent response to application of ATP is suppressed by inhibition of tissue-nonspecific alkaline phosphatase, highlighting the significance of this enzyme for the breakdown of ATP in the olfactory bulb [22]. The EC50 for adenosine to elicit Ca2+ signalling via A2A receptors in olfactory bulb astrocytes is less than 1 μM, while the EC50 for Ca2+ signalling upon ATP- and ADP-mediated P2Y1 receptor stimulation is higher by more than one order of magnitude [22]. Hence, astrocytes respond to adenosine derived from low amounts of ATP only via A2A receptor activation, whereas large amounts of ATP also stimulate P2Y1 receptors. Stimulation of P2Y1 and A2A receptors results in the activation of phospholipase C and subsequent production of IP3 which releases Ca2+ from internal stores. This Ca2+ increase is enhanced by the Ca2+-dependent stimulation of the IP3 receptor, a mechanism termed Ca2+-induced Ca2+ release [23]. In addition, Ca2+ store depletion upon sustained stimulation of P2Y1 receptors activates store-operated Ca2+ channels in the plasma membrane, leading to persistent Ca2+ influx in order to refill depleted Ca2+ stores [109]. The resulting cytosolic Ca2+ increase spreads throughout the astrocytic syncytium within a glomerulus, but does not cross the border to adjacent glomeruli,


since only astrocytes sending processes into the same glomerulus are coupled by gap junctions [102]. In the olfactory bulb, stimulation of astrocytes has been shown to trigger the release of glutamate and GABA from astrocytes, evoking long-lasting de- or hyperpolarizations of mitral and granule cells [65]. Hence, astrocytes balance the neuronal excitability, in particular, glomeruli and modulate network activity within the olfactory bulb. Mitral cells themselves respond to P2Y1 receptor activation with an increase in excitation [31], and the combination of astrocytic and neuronal purinergic signalling in the glomeruli provides a complex machinery to adjust olfactory information processing in the olfactory bulb. Purinergic signalling in olfactory ensheathing cells The first study to show physiological functions of purinergic signalling in the olfactory bulb was performed on OECs [99]. OECs express P2Y1 receptors and respond to application of ATP with calcium increases (Fig. 3c). Adenosine receptors appear not to be linked to calcium signalling in OECs, in contrast to olfactory bulb astrocytes [22, 99]. A recent study demonstrated that purinergic calcium signalling does not occur in all OECs in the olfactory bulb. Rather, only P75positive OECs in the outer nerve layer respond to ATP or other purinergic ligands, whereas P75-negative OECs in the inner nerve layer do not generate calcium signals [116]. This spatial heterogeneity in neurotransmitter sensitivity seems to be a general phenomenon, since OECs in the inner nerve layer were inert to other canonical neurotransmitters as well, while OECs in the outer nerve layer also responded to acetylcholine, noradrenaline, serotonin, dopamine and glutamate [116]. Interestingly, glutamate-evoked calcium responses in this subpopulation of OECs is mediated by mGluR1, a metabotropic glutamate receptor preferentially expressed in neurons, while in astrocytes of the olfactory bulb as well as in other brain areas, mGluR5 is found [11, 93, 106, 123]. Like in astrocytes, neurotransmitter-mediated Ca2+ transients in OECs are fostered by Ca2+-induced Ca2+ release via InsP3 receptors and prolonged by store-operated Ca2+ entry [111, 116]. The close morphological and functional relationship between olfactory sensory axons and OECs renders the system ideal to study the mechanism(s) of transmitter-mediated neuron-glia interactions. OECs in the outer nerve layer monitor the release of glutamate and ATP and can respond with a calcium increase even to a single compound action potential in an axon bundle (unpublished observation). The release of both ATP and glutamate from receptor axons is calcium dependent and accompanied by vesicle fusion [115]. When refilling of synaptic vesicles is impaired by bafilomycin A1, or vesicle fusion is suppressed by botulinum toxin, electrical stimulation of receptor axons fails to elicit calcium signalling in OECs, unequivocally demonstrating the exocytotic nature of ATP (and glutamate) release from olfactory receptor neurons not

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only at synaptic terminals, but also along the axons [74, 115]. This system was also employed to estimate the concentration of extracellular ATP released by receptor axons. Action potentials in a bundle of olfactory sensory axons evoked the release of ATP that mediated ionic currents in P2X 2 transfected HEK cells (‘sniffer cells’) used as biosensors [115]. The size of the current evoked by a brief burst of ten action potentials matched the size of a current evoked by 100 μM ATP directly applied from a micropipette to the sniffer cell. Hence, the concentration of extracellular ATP released from neurons may reach the upper micromolar range and, thus, is sufficient to saturate P2Y or adenosine receptors of postsynaptic neurons and glial cells. Other types of sensory neurons such as dorsal root ganglia (DRG) cells also release ATP by exocytosis, which results in calcium signals in satellite glial cells [147]. They possess ATP-containing vesicles that comprise VNUT, suggesting that vesicular release of ATP is the main purinergic pathway from DRG cells to glial cells [54]. This may also apply to olfactory bulb neurons, since VNUT is also highly expressed in the olfactory bulb [69].

Neurovascular coupling in the retina and olfactory bulb In several brain areas, glial cells have been shown to translate neuronal activity into vascular responses, a mechanism termed

Fig. 4 Role of purinergic signalling for neurovascular coupling in the olfactory bulb. Periglomerular astrocytes respond to ATP and adenosine with Ca2+ signalling. The increase in cytosolic Ca2+ can evoke both constriction and dilation of adjacent arterioles and capillaries in the glomerular layer, but also of arterioles in the nerve layer. In contrast, capillaries in the nerve layer are in contact with OECs, and Ca2+ increases in OECs mediate vasoresponses in capillaries

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neurovascular coupling [41]. In general, glial cells detect neural activity via synaptically released neurotransmitters and respond with calcium signalling which, in turn, results in the release of vasoactive substances such as prostaglandin E2 (PGE2) [2, 92]. In the retina, the calcium increase in Müller glial cells resulting from neuronal ATP release triggers the secretion of PGE2 and epoxyeicosatrienoic acid [80, 86]. These substances lead to a dilation of adjacent arterioles and hyperaemia. However, under conditions of high O2 or high NO concentrations, calcium signalling in Müller glial cells has been shown to result in a vasoconstriction, attributable to the release of 20-hydroxyeicosatetraenoic acid from glial cells [80, 81]. A recent in vivo study implicates that the vascular tone of arterioles in the rat retina is maintained by the tonic release of ATP most likely acting on P2X1 receptors; aside from neurons, glial cells have been identified as putative sources of ATP in these experiments [67]. Hence, the blood flow through retinal blood vessels is controlled by the release of ATP from both neurons and glial cells, as well as by the metabolic state of the tissue. In the olfactory bulb, neurovascular coupling is mediated by both astrocytes and OECs (Fig. 4) [74]. Calcium signalling in periglomerular astrocytes is followed by dilation and constriction, respectively, of arterioles that penetrate the pia mater plus the nerve layer and are covered by astrocytic endfeet [23, 93]. In isolated olfactory bulbs in toto, Ca2+ signalling in astrocytes evoked a vasoconstriction [23]. In contrast, odor stimulation of olfactory receptor neurons in vivo in anaesthetized mice always resulted in a vasodilation and an increase in cerebral blood flow [93], suggesting that the vasoconstriction found in the in situ preparation is due to artificial conditions such as high O2 concentration in the experimental saline and lack of vascular tone. When astrocytic mGluR5 receptors and glutamate transporters were blocked in vivo, the increase in blood flow evoked by odor stimulation was greatly reduced [93]. The remaining increase in blood flow is not attributable to glutamate but might be mediated by ATP, since ATP is coreleased from olfactory sensory axons upon stimulation and excites astrocytes [22]. While arterioles penetrating the olfactory bulb nerve layer are covered by astrocytic endfeet, capillaries branching off these arterioles and supplying the nerve layer lack GFAP-positive astrocytic elements [23]. Rather, they are encompassed by processes of OECs [115]. Stimulation of olfactory sensory axons in an in toto preparation of the olfactory bulb evoked calcium signalling in OECs that was accompanied by the constriction of adjacent capillaries [115]. Blocking P2Y1 receptors not only reduced the calcium responses in OECs, but also suppressed vasoconstriction, demonstrating that OEC-mediated neurovascular coupling between olfactory sensory neurons and nerve layer capillaries (at least partly) depends on purinergic signalling.


Conclusion Purinergic signalling is an important mediator of neuron-glia communication in sensory systems contributing to tissue homeostasis and to information processing. This includes volume regulation, neurovascular coupling, synaptic modulation and regulation of development. Since both glial physiology and purinergic neurotransmission are rapidly evolving fields, one can expect that more functional interactions of glial cells with neurons will be uncovered. This will elucidate the impact of glial cells on neuronal performance, and the modulatory actions of ATP and adenosine, in the nervous system in general and particularly in the transduction and processing of sensory stimuli. Acknowledgments Financial support by the Deutsche Forschungsgemeinschaft (LO779/3 and GRK845 to C.L.; LO779/6 to C.L. and D.H.; RE849/12, RE 849/16, GRK 1097 and SPP 1172 to A.R.; FOR 748 to A.G. and A.R.; GR4403/1-1 to A.G.), the EU FP 7 Program EduGlia 237956 to A.R. and the PRO RETINA-Stiftung to A.G. is gratefully acknowledged.

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