Bergmann Glial Cells

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Bergmann Glial Cells A Verkhratsky, The University of Manchester, Manchester, UK A Reichenbach, University of Leipzig, Leipzig, Germany ã 2009 Elsevier Ltd. All rights reserved.

The cerebellum of all vertebrates contains specialized radial astrocytes, which send long, vertically oriented processes toward the pia. These glial processes were discovered by Karl Bergmann in 1857 as cerebellar radial fibers. In 1871, Camillo Golgi recognized these structures as cells and found their somata/cell bodies, from where the radial fibers originated. Both Golgi and Ramon-y-Cajal referred to these cells as epithelial cells, and for many years they were generally known as Golgi epithelial cells. In recent times, the common name for these cerebellar radial astrocytes is Bergmann glial cells.

Morphology The somata of Bergmann glial cells are localized in the Purkinje cell layer (Figure 1). Each Bergmann glial cell has three to six stem processes, which protrude through the molecular layer toward the pial surface. These processes contain many mitochondria and bundles of intermediate filaments, constituted by vimentin and glial fibrillary acidic protein (GFAP). They can be labeled by antibodies directed against these proteins, which reveals that they are organized in palisades, parallel to the long axis of each folium. On average, every Bergmann glial cell in rodent cerebellum has a volume of approximately 3600 mm3, and altogether these cells occupy approximately 15–18% of the volume of the molecular layer. There are approximately eight Bergman glial cells per every Purkinje neuron; the processes of the former develop intimate relations with the dendritic arborization of the latter. The stem processes of Bergmann glial cells end at the pia mater, with so-called endfeet, which together form a complete covering of the cerebellar surface. The subpial endfoot membranes are endowed with orthogonal arrays of membrane particles, probably representing locally enriched membrane proteins such as the water pore channel, aquaporin-4, and related channel and transporter proteins devoted to an active exchange of ions and water between the neuropile and the subarachnoidal space. On their way from soma to the pia, the stem processes extend numerous side branches, which form close contacts with synapses present on the dendrites of Purkinje neurons; on average, every Bergmann glial cell covers 2000–6000 such synapses.

Morphologically, these gliosynaptic contacts are quite peculiar because they are organized in the form of morphofunctional microdomains (Figures 2 and 3). The microdomains are constructed by protrusions sent from the stem process at rather regular intervals. Each microdomain is made of a thin stalk and a complex ‘head’ structure, which in turn forms a lamellar perisynaptic sheath for approximately five synapses. The stalk is extremely thin (
0.3 mm) and long (up to 7 mm); the head is a complex structure with an extremely large surface area (
300 mm2) formed by a relatively small (
16 mm3) volume. The surfaces of the appendages may form the major part of Bergmann glial plasmalemma (overall, the surface-to-volume ratio for Bergmann glial cell varies between 13 and 17 mm1, whereas the same ratio for appendage may reach 25 mm1). The synaptic–glial interactions, therefore, are very much confined to these single microdomains; and these microdomains can be independently excited. Each microdomain also contains on average two mitochondria, which may provide a local energy supply and also serve as a barrier preventing Ca2þ diffusion. The coverage of synapses by glial membranes is quite specific because glial membrane tongues almost completely seal the synaptic clefts while leaving a considerable part of extrasynaptic structures uncovered.

Physiology Membrane Conductance

The resting membrane potential of mature Bergmann glial cells is between 80 and 90 mV, and the resting input resistance is quite low (40–100 mO). Potassium channels are responsible for the major part of membrane permeability of Bergmann glial cells. The expression of these channels is developmentally regulated. In the early postnatal (< postnatal day 7 (P7)) period, Bergmann glial cells demonstrate a mixture of inwardand outward-rectifying potassium currents, which can be blocked by cesium, barium, and 4-aminopyrindine. Mature Bergmann glial cells (> P20) display a large passive Kþ conductance manifested by voltage- and time-independent Kþ currents, insensitive to Csþ, Baþ, or 4-AP. In addition, these cells have a complement of inward- and outward-rectifying Kþ channels but their density is quite low. There were some reports that Bergmann glial cells express mRNA for the voltage-gated Naþ channel Nav1.6, but functional channels were never detected electrophysiologically. Gap Junctions

Similar to other astrocytes, Bergmann glial cells are coupled by gap junctions. The main gap junction

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d Figure 1 Morphology of Bergmann glial cells in the vertebrate cerebellum. (a–c) Bergmann glial cells of the adult rat cerebellum, immunocytochemistry. The glutamine synthetase-positive Bergmann glial cell somata (dark) surround and envelop the Purkinje cell somata (a and b), while their glial fibrillary acidic protein (GFAP)-positive stem processes run through the molecular layer where they form into rows or palisades (view from the cerebellar surface; c). (d–f) Camera lucida drawings of Golgi-stained Bergmann glial cells in the cerebella of several different mammals, including those of human (d), the trout Salmo gairdneri (e), and the chicken (f). Adapted from Reichenbach A and Wolburg H (2005) Astrocytes and ependymal glia. In: Kettenmann H and Ransom BR (eds.) Neuroglia, pp. 19–35. Oxford: Oxford University Press.

protein expressed by Bergmann glial cells is connexin43x. The gap junction coupling (as was judged by both electrophysiological recordings and dye coupling; Figure 4) between Bergmann glial cells begins to appear at approximately P7 and is fully developed by P20. The gap junctions are preferentially localized in the distal processes of Bergmann glial cells. Dye spread between Bergmann glial cells in cerebellar slices revealed a peculiar organization of the glial syncytium: dye-coupled cells were always arranged in rows perpendicular to parallel fibers (Figure 4). This is very

similar to the two-dimensional orientation of Purkinje neurons and, therefore, both neuronal and glial elements from cellular stacks, which may also have functional impact. There are also indications for the existence of gap junction coupling between Bergmann glial cells and adjacent Purkinje neurons. Neurotransmitter Receptors

Glutamate receptors The main type of ionotropic glutamate receptors expressed by Bergmann glial cells is of the a-amino-3-hydroxy-5-methyl-4-isoxazole

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Figure 2 Bergmann glial microdomains. (a) Reconstruction of an appendage based on electron microscopic data. (1) Fluorescence light micrograph of a dye-injected Bergmann glial cell is shown; the red square (20  20 mm) corresponds to the portion that was reconstructed from consecutive ultrathin sections. (2) One of the lateral appendages, arising directly from fiber with all the other side branches omitted for clarity. (3) The same appendage as shown in 2, but with one of the appendages marked by blue. This labeled structure is shown in isolation and at higher magnification in (b). (b) Fine structure of appendages and relation to synapses. (Left) A small lateral appendage, arising from the reconstructed part of the glial fiber (blue in a3), is shown as a slightly turned, isolated three-dimensional reconstruction. Electron micrographs of four sections contributing to the reconstruction (designated 1–4) are shown on the right; glial compartments appear black after conversion of the injected dye. The location of these sections in the reconstruction is indicated by the labeled arrows. (1) Region directly contacting synapses. (2) Glial compartments without direct synaptic contacts. (3) Bulging glial structure containing a mitochondrion. (4) The stalk of the appendage. (c) Another example of a synapse contacted by glial compartments, appearing as black structures (white arrows); in this case, the postsynaptic element can be traced back along the spine (black arrows) to the Purkinje cell dendrite (reddish overlay; the presynaptic terminal is labeled by a green overlay). Adapted from Grosche J, Matyash V, Moller T, Verkhratsky A, Reichenbach A, and Kettenmann H (1999) Microdomains for neuron–glia interaction: Parallel fiber signaling to Bergmann glial cells. Nature Neuroscience 2: 139–143.

propionic acid (AMPA) subtype. Bergmann glial cells do not possess the glutamate receptor (GluR)2 subunit, making their AMPA receptors Ca2þ permeable. Indeed, activation of AMPA receptors by exogenous kainate triggers substantial Ca2þ rises in glial cells (Figure 5). There are indications that Bergmann glial cells may express mRNA for the NR2B subunit of N-methyl-D-aspartate (NMDA) receptors; application of endogenous NMDA was reported to trigger [Ca2þ]i elevation and small inward currents. These

responses, however, were often blocked by tetrodotoxin, and thus most likely resulted from stimulation of neurons. Bergmann glial cells express metabotropic glutamate receptors of the mGluR5 type; stimulation of these receptors triggers an inositol 1,4,5-triphosphate (InsP3)-mediated release of Ca2þ from the endoplasmic reticulum (ER) intracellular Ca2þ stores. This mGluR5dependent Ca2þ mobilization assumes the leading role in the generation of glial Ca2þ signals following

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Figure 3 Glial microdomains form a substrate for glial–synaptic interactions. (a) Three-dimensional reconstruction of a group of neighboring cerebellar synapses (yellow; synaptic clefts: orange) together with the surrounding leaflets provided by the labeled Bergmann glial cell (blue-green). Arrowheads point to neuronal surfaces not covered by glial sheaths from the labeled cell. (b) Schematic representation of a Bergmann glial microdomain. The basic components of the microdomain, the stalk and the ‘head,’ are shown together with their relationships to the neighboring neuronal elements. Stimulation of several closely positioned parallel fibers may activate a single microdomain, inducing both membrane currents and local Ca2þ signals. Adapted from Grosche J, Kettenmann H, and Reichenbach A (2002) Bergmann glial cells form distinct morphological structures to interact with cerebellar neurons. Journal of Neuroscience Research 68: 138–149.

stimulation with glutamate. The contribution of the AMPAR-mediated Ca2þ influx is much smaller because of rapid desensitization of these receptors in the presence of glutamate. Nonetheless, the AMPA receptor-mediated Ca2þ entry is functionally important because adrenoviralassisted genetic introduction of the GluR2 subunit into Bergmann glial cells extinguished the Ca2þ permeability of these receptors and resulted in a withdrawal of glial processes from the synapses formed on Purkinje neurons. This, in turn, affected synaptic transmission by slowing the glutamate uptake, and it promoted the reinnervation of Purkinje neurons by several climbing fibers. GABA receptors GABAA ionotropic receptors are prominently expressed in young Bergmann glial cells; application of g-aminobutyric acid (GABA) triggers

large Cl currents in cells from P5 to P12 animals. Immunohistochemical techniques revealed a prominent expression of a2, a3, and g subunits in these cells. The GABAA receptors are predominantly located in the distal processes of the Bergmann glial cells. The expression of GABAA receptors is downregulated during postnatal development; by P20, the amplitudes of GABA-induced currents are very much diminished and only a2 and g1 subunits remain detectable immunohistochemically. The GABAA receptors in mature Bergmann glial cells are primarily concentrated in the parts of glial membrane ensheathing inhibitory GABAergic synapses formed on Purkinje neurons. In addition to GABAA receptors, immunoreactivity for GABAB1 receptors was detected in Bergmann glial cells; there are also some indications for the possible expression of GABACRs. The functional significance of these receptor types remains to be explored.

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Figure 4 Gap junction coupling of Bergmann glial cells. (a) Dye coupling. Bergmann glial cell was dialyzed via the patch pipette with a solution containing Lucifer yellow. Fluorescence images were recorded a t times indicated times after the whole cell configuration was established using confocal microscopy. Images were three-dimensional reconstructed and illustrate the spread of dye to adjacent, coupled cells. The number of coupled cells are as follows: 1, n ¼ 4; 2, n ¼ 8; 3, n ¼ 24; 4, n ¼ 26; 5, n ¼ 26. (a6) The image shown in a5 is rotated to the parasagittal orientation. The upper 25 pm of the 120 pm thick cerebellar slice is displayed. (b) Schematic image showing orientation of the coupled Bergmann glia cells. In the parasagittal sections used, the array of coupled cells extends parallel to the slice surface and perpendicular to the parallel fibers. BG, Bergmann glia cells; PC, Purkinje cell. Adapted from Muller T, Moller T, Neuhaus J, and Kettenmann H (1996) Electrical coupling among Bergmann glial cells and its modulation by glutamate receptor activation. Glia 17: 274–284.

Purinoreceptors Bergmann glial cells respond to extracellular application of ATP by robust cytosolic Ca2þ rises, which are initiated in the processes and then spread toward the soma (Figure 6). These ATPinduced Ca2þ responses are mediated by P2Y metabotropic purinoreceptors and subsequent InsP3-induced Ca2þ release from the ER Ca2þ stores. This conclusion is based on the following observations: (1) ATP-induced Ca2þ signals are preserved in Ca2þ-free extracellular solutions; (2) applications of ATP do not trigger any measurable membrane currents; and (3) ATP-induced Ca2þ signaling is inhibited either by the ER calcium pump blocker, thapsigargin, or by intracellular administration of the competitive InsP3 receptors antagonist, heparin. Incidentally, the P2Y purinoreceptors in Bergmann glial cells can be activated by extracellular administration of NAADP. Adrenoreceptors Application of endogenous adrenomimetics (epinephrine and norepinephrine) induces cytosolic Ca2þ elevations in Bergmann glial cells.

These Ca2þ responses are of purely metabotropic (G-protein/phospholipase C (PLC)/InsP3-mediated) nature; they are unaffected by Ca2þ removal from extracellular milieu and blocked by thapsigargin or intracellularly administered heparin. The monoamine-induced Ca2þ signaling is mediated through a1 adrenoreceptors; the Ca2þ responses can be mimicked by the specific a1 adrenoreceptor agonist, phenylephrine, but not by b-adrenomimetic isoproterenol or a2-adrenomimetic clonidine. Moreover, the monoamine-evoked Ca2þ signals can be almost completely blocked by the a1 adrenoreceptor antagonist prazozin; however, they are fully resistant to the action of the a2 adrenoreceptor antagonist yohimbine or the b adrenoreceptor antagonist propranolol.

Histamine receptors Bergmann glial cells express functional H1 histamine receptors. Stimulation of these receptors triggers InsP3-induced Ca2þ release from the ER. Histamine-induced Ca2þ signaling is

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b Figure 5 Stimulation of ionotropic glutamate receptors (GluRs) in Bergmann glial cell in cerebellar slices triggers Ca2þ influx. (a) Pseudocolor image of a fura-2-loaded Bergmann glial cell (a scheme of the experiment is shown on the right) in control conditions and upon extracellular application of 100 mM kainate. Note preferential increase in [Ca2þ]i in cell processes. (b, c) [Ca2þ]i and membrane current traces recorded from the same cell. Both removal of extracellular Ca2þ (b) and blockade of ionotropic GluRs by 6-cyano-7 nitroquinoxaline-2,3-dione (CNQX; 10 mM; c) inhibited kainate-induced currents and [Ca2þ]i elevation, suggesting a key role for Ca2þ influx. F340/380, fluorescence ratio at 340/380 nm. Adapted from Verkhratsky A, Orkand RK, and Kettenmann H (1998) Glial calcium: Homeostasis and signaling function. Physiological Reviews 78: 99–141.

very substantially inhibited by the selective H1 antagonist chlorpheniramine. Endothelin receptors All three forms of endothelin, ET-1 to ET-3, when applied extracellularly trigger Ca2þ signals in Bergmann glial cells. The receptors responsible are selectively sensitive to the ETB receptor agonist BQ3020 and are blocked by the ERB receptor antagonist BQ-788; the expression of ETB receptors was further confirmed by single-cell polymerase chain reaction. Intracellularly, the stimulation of ETB receptors results in activation of the PLC/ InsP3/Ca2þ release cascade. Bergmann glial cells in situ express a defined set of neurotransmitter receptors Studies on Bergmann glial cells in situ (i.e., in cerebellar slices) revealed that they are always endowed with a strictly defined pattern of functional neurotransmitter receptors: all cells were sensitive to glutamate, ATP, GABA,

monoamines, histamine, and endothelin. By contrast, many other neuroactive substances known to activate cultured astrocytes (e.g., bradykinin, oxytocin, substance P, adenosine, vasopressin, or PAF) did not affect the cells. Most interestingly, the sensitivity of Bergmann glial cells to neurotransmitters is very similar to that of its closest neighbor, the Purkinje neuron: in general, the modalities of receptors expressed by these two cell types are identical. Furthermore, neurotransmitter receptors expressed by the neuron and by the glial cell specifically match the neurotransmitters released in their vicinity (Figure 7), thus allowing them to sense the very same incoming information. Neurotransmitter Transporters

Glutamate transporters Membranes of Bergmann glial cells, surrounding the excitatory glutamatergic synapses formed on the dendrites of Purkinje neurons, contain the glutamate transporter GLAST (glutamate aspartate transporter) and GLT-1 (the former being the

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Figure 6 ATP-induced Ca signaling in Bergmann glial cells results exclusively from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2þ release from ER Ca2þ stores. (a) ATP-induced [Ca2þ]i transients were measured from ‘bulk-loaded’ Bergmann glial cells stained by incubating cerebellar slices in fura-2 acetoxymethyl ester (AM)-containing solutions. Addition of ATP triggered an increase in [Ca2þ]i that persisted in Ca2þ-free extracellular solution. (b) In a similar experiment, incubation of slice with 500 nM thapsigargin completely and irreversibly blocked ATP-induced Ca2þ signaling. (c) Intracellular administration of heparin via intracellular dialysis with a patch pipette inhibited [Ca2þ]i increase induced by ATP. Control [Ca2þ]i transient was recorded from fura-2 AM-loaded cells before commencing intracellular dialysis. (d) Illustration of spatial distribution of [Ca2þ]i at the time of maximum ATP response. Note the higher levels of [Ca2þ]i in Bergmann glial cell processes compared with the cell body. Adapted from Kirischuk S, Moller T, Voitenko N, Kettenmann H, and Verkhratsky A (1995) ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells. Journal of Neuroscience 15: 7861–7871; and Verkhratsky A, Orkand RK, and Kettenmann H (1998) Glial calcium: Homeostasis and signaling function. Physiological Reviews 78: 99–141.

dominant type), whereas the extrasynaptic regions of the neuronal (i.e., Purkinje cell) spines are rich in neuronal excitatory amino acid transporters of the EAAT4 type. The glial and neuronal transporters play distinct roles in controlling the glutamate concentration within and around the synaptic cleft. The affinity of GLAST to glutamate is approximately 10 times lower than that of excitatory amino acid transporter 4 (EAAT4); therefore, GLAST is primarily responsible for rapid uptake of glutamate following its exocytosis. This uptake determines the time course of synaptic events and prevents an escape of high glutamate concentrations from the cleft. Inhibition of GLAST significantly increases the early spillover of glutamate, as judged, for example, by the activation of NMDA receptors on GABAergic terminals, with a subsequent increase in the strength of inhibitory input to Purkinje neurons. In this way, glutamate uptake by Bergmann glial cells can regulate the synaptic inhibition of Purkinje neurons. Furthermore, inhibition of glial glutamate transporters also affects background synaptic transmission to Purkinje

neurons by increasing the frequency of spontaneous miniature excitatory postsynaptic currents (EPSCs). The EAAT4, in contrast, buffers low glutamate concentrations and prevents the delayed spillover of the neurotransmitter to neighboring synapses. According to various estimates, approximately 80% of glutamate released during physiological neurotransmission is accumulated by Bergmann glial cells and only 20% is removed by the neuronal postsynaptic compartment. To translocate glutamate against the concentration gradient, both neuronal and glial transporters utilize transmembrane gradients of Naþ and Kþ. Uptake of a single glutamate molecule requires influx of three Naþ ions and efflux of one Kþ ion; in addition, glutamate brings one more Hþ ion into the cell (the net cation influx underlies the electrogenic role of transporters). Therefore, the maintenance of transmembrane ion gradients is essential for an effective performance of glutamate transporters. Exposure of Bergman glial cells to glutamate triggers substantial increases in the cytoplasmic Naþ concentration, which may impair further glutamate uptake. This

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BA ST Figure 7 Bergmann glial cell and its neighbor Purkinje neuron bear a similar set of neurotransmitter receptors. NT, terminals from tuberomammillary nucleus of the posterior hypothalamus that carry histamine innervation of the cerebellar cortex; LC, terminals from locus coeruleus that utilize noradrenalin and ATP as neurotransmitters; CF and PF, climbing and parallel fibers, respectively, major neurotransmitter glutamate; BA and ST, basket and stellate cells that deliver GABA to the Purkinje neuron layer. Adapted from Verkhratsky A, Orkand RK, and Kettenmann H (1998) Glial calcium: Homeostasis and signaling function. Physiological Reviews 78: 99–141.

[Naþ]i increase can be counteracted by a sodium– calcium exchanger, expressed in the membrane of Bergmann glial cells; sharp cytosolic Naþ rises turn the sodium–calcium exchanger into the reverse mode (when Naþ is expelled in exchange for Ca2þ). This process may rapidly lower [Naþ]i and thus sustain the operation of the glutamate transporter. Under pathological conditions that are accompanied by cell depolarization and accumulation of glutamate and Naþ ions in the glia, the glutamate transporter may also reverse and start to extrude glutamate from the Bergman glial cells, thus exacerbating the glutamatedependent excitotoxic neuronal death. In addition, Bergmann glial cells express functional cysteine/ glutamate transporters, which also may provide a pathway for glutamate extrusion during periods of ischemia. GABA transporters Bergman glial cells are endowed with a specific GABA uptake system, represented by high-affinity GABA/Naþ/Cl transporters of the GAT-1 type. The uptake of one GABA molecule is coupled to the inward transport of two Naþ ions and

one Cl ion. This transporter is present in the Bergmann glial cell somata and is also concentrated in the processes enwrapping GABAergic synapses formed on Purkinje neurons. In these compartments, glial GABA uptake can be relevant for the neurotransmitter removal during normal synaptic activity. Under experimental conditions, when the Bergmann glial cells were internally perfused with solutions containing 10 mM GABA and 12.5 mM Naþ, cell membrane depolarization to 40 mV resulted in the reversal of the GABA transporter. Whether such a reversed GABA transport may be relevant in vivo (even under pathological conditions) remains unclear. Glycine transporters Bergmann glial cells possess functional GlyT1 transporters that belong to the Naþ/Cl-dependent transporter family. These transporters can work in both forward (GABA uptake) and reverse (GABA extrusion) modes, and under certain conditions they can execute glycine efflux. However, this requires a depolarization of the Bergmann glial cell to þ20 mV, which is very unlikely to occur under physiological conditions.

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Taurine transporters Bergmann glial cells express a Naþ/Cl-dependent taurine transporting system, which is able to function in both forward (taurine uptake) and reverse (taurine release) modes. The reversal of the transporter requires cell depolarization (up to 50 mV) together with an increase in the intracellular Naþ concentration above 40 mM; these conditions most likely may occur only during severe ischemia. Functionally, the taurine transporter (working in the forward mode) could be involved in taurine redistribution following a hypo-osmotic shock. Responses of Bergmann Glial Cells to Neuronal Activity

Electrical responses Stimulation of synaptic inputs to Purkinje neurons, formed by parallel and climbing fibers, induces complex membrane current responses in Bergmann glial cells. These current responses are mediated by activation of both ionotropic AMPA-type glutamate receptors and Naþ-dependent glutamate transporters. The glial AMPA receptor-mediated responses show short-term and long-term plasticity. The former is manifested by prominent paired-pulse facilitation, whereas the latter appears as long-term depression of synaptically induced glial responses following low-frequency repetitive stimulation of parallel fibers. These plasticity patterns may allow Bergman glial cells to distinguish between synaptic inputs displaying different activities. In addition, Bergmann glial cells can also be activated by ectopic glutamate release from climbing fibers. This ectopic release predominantly activates glial AMPA receptors, and it may be important for rapid communication between nerve afferents and Bergmann glial cells. Interestingly, direct electrical stimulation of Bergmann glial cells was reported to affect the ‘background’ synaptic activity between parallel fibers and Purkinje neurons. Fifty consecutive depolarizations of Bergmann glial cells (0.5 s duration, delivered at 1 Hz) substantially reduced the frequency of spontaneous postsynaptic currents recorded from the neuron. Cytoplasmic calcium responses In parallel with membrane currents, electrical stimulation of parallel fibers triggers cytosolic calcium responses in the Bergmann glia. These Ca2þ responses are precisely localized, being limited to small compartments within glial cell processes (Figure 8). The size of these Ca2þ response sites is similar to that of morphologically distinct microdomains, which enwrap a cluster of synapses formed by parallel fibers. This striking compartmentalization of synaptically induced glial Ca2þ responses may be important for spatial discrimination of the incoming signals. The Ca2þ responses elicited in Bergmann glial cells following nerve activity

result from activation of several pathways, including activation of glutamate receptors and a1 adrenoreceptors – with subsequent Ca2þ release from the ER stores – as well as the NO signaling cascade, which activates plasmalemmal Ca2þ influx. The local Ca2þ signals in the Bergmann glial processes also occur spontaneously, without stimulation of synaptic inputs; most likely, this is the glial correlate of spontaneous EPSCs in nerve cells.

Development and Morphogenesis of Cerebellar Structures During ontogenesis, Bergmann glial cells arise by transformation (i.e., loss of the ventricular cell process) from fetal radial glial cells, as well as by proliferation of immature Bergmann glial cells within the primitive molecular layer up to the second postnatal week in rat. From the beginning of their existence, Bergmann glial cells are intimately involved in cerebellar morphogenesis. First, in the early postnatal period (P4–P15) Bergmann glial cells act as a guidance system for young postmitotic granule cells, which migrate from the proliferative zone located on the surface of the cerebellar cortex (external granule cell layer) to the inner granule cell layer positioned below the Purkinje cell bodies. This migratory process involves several defined morphological stages described in detail by Ramon-y-Cajal and Pasko Rakic. First, the newborn granule neuron contacts the fiber of a Bergmann glial cell located within the deep part of the external granule layer. After establishing such a contact, the granule neuron turns first into a bipolar cell and then extends a third process that becomes the leading one, guiding the bipolar neuron along the Bergmann glial cell fiber. When this leading process elongates sufficiently, the soma (including the nucleus) of the granule neuron translocates through it, such that finally the entire neuron attains its destination. Starting from the third postnatal week, Bergman glial cells become closely associated with Purkinje neurons. This association appears to be reciprocal because without contact with Purkinje cells, the Bergmann glia cannot develop its mature phenotype, whereas the development of Purkinje cell dendrites occurs in close alignment with Bergmann glial cell processes. These developmental neuronal–glial interactions essentially involve the Notch signaling system. Membranes of Purkinje cell dendrites express specific Delta/Notch-like EGF-related receptors (DNER), whereas the processes of Bergmann glial cells express Notch. Binding of DNER to Bergmann glial processes determines the morphological differentiation of the latter, and the inhibition of this type

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b Figure 8 Stimulation of parallel fibers triggers local calcium signals in Bergmann glial cells. (a) Experimental protocol. Parallel fibers (PF) were stimulated via a pipette connected to a stimulator (STIM) while calcium-dependent fluorescence responses were recorded in a Bergmann glial cell (BG). PCL, Purkinje cell layer. (b) Confocal fluorescence intensity image of a patch-clamped Bergmann glial cell dialyzed with the calcium indicator, Oregon green 488 BAPTA-1 (center right). Three processes were distinguished (indicated as 1–3). Calcium signals in response to PF stimulation were measured independently for each process (left). Time of PF stimulation is indicated by arrows. The responding process (1) was then further subdivided into five regions of interest, in which calcium signals were measured separately (right). Time of PF stimulation is indicated by an arrow and a dotted line. The center left image was obtained from a sequence of serial sections and shows the cell in depth (turned by 90 compared to the right image). The lines indicate the focal plane used for [Ca2+]i recordings. The process is thus within the volume from which recordings were obtained. Adapted from Grosche J, Matyash V, Moller T, Verkhratsky A, Reichenbach A, and Kettenmann H (1999) Microdomains for neuron–glia interaction: Parallel fiber signaling to Bergmann glial cells. Nature Neuroscience 2: 139–143.

of local signaling prevents normal morphogenesis of the glial cells. The intermediate filaments in the glial cell processes may also play a crucial role in these glial–neuronal interactions; in vimentin-deficient mice, many of the Purkinje neurons were found to display a paucity of spiny branchlets, a reduced number of synaptic inputs, and degenerative alterations in their cytoplasm.

Conclusions Bergmann glial cells are specialized radial astrocytes that constitute an important cellular element of cerebellar architecture. These cells control the

development of cerebellar circuits by guiding the granule cells from the external to the internal granule cell layer, and they assist in the morphogenesis of Purkinje neurons. Mature Bergmann glial cells form complex ‘microdomain’ structures that ensheath the synaptic contacts formed on the dendrites of Purkinje cells. Functionally, Bergmann glial cells are endowed with a well-defined set of neurotransmitter receptors, which allow them to sense the activity of synapses formed on the Purkinje neurons. Neuronal activity stimulates the receptors localized in the Bergmann glial cell membrane, resulting in an activation of both transmembrane currents and intracellular Ca2þ responses. The latter are highly localized within the

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microdomains, thus enabling spatial discrimination of the incoming signals. Bergmann glial cells also possess numerous transporter systems that participate in the clearance of neurotransmitters released during synaptic activity. Thus, Bergmann glial cells can be regarded as active elements of information processing in the cerebellum. See also: Astrocyte: Response to Injury; Cerebellum: Clinical Pathology; Cerebellum: Evolution and Comparative Anatomy; Enteric Nervous System: Glial Cells and Interstitial Cells of Cajal; Gamma-Aminobutyric Acid (GABA); Glial Cells: T Cell Interactions; Glial Glutamate Transporters.

Further Reading Bellamy TC and Ogden D (2005) Short-term plasticity of Bergmann glial cell extrasynaptic currents during parallel fiber stimulation in rat cerebellum. Glia 52: 325–335. Bordey A and Sontheimer H (2003) Modulation of glutamatergic transmission by Bergmann glial cells in rat cerebellum in situ. Journal of Neurophysiology 89: 979–988. Burnashev N, Khodorova A, Jonas P, et al. (1992) Calciumpermeable AMPA-kainate receptors in fusiform cerebellar glial cells. Science 256: 1566–1570. Clark BA and Barbour B (1997) Currents evoked in Bergmann glial cells by parallel fibre stimulation in rat cerebellar slices. Journal of Physiology 502: 335–350. Grosche J, Kettenmann H, and Reichenbach A (2002) Bergmann glial cells form distinct morphological structures to interact with cerebellar neurons. Journal of Neuroscience Research 68: 138–149. Grosche J, Matyash V, Moller T, Verkhratsky A, Reichenbach A, and Kettenmann H (1999) Microdomains for neuron–glia interaction: Parallel fiber signaling to Bergmann glial cells. Nature Neuroscience 2: 139–143. Huang H and Bordey A (2004) Glial glutamate transporters limit spillover activation of presynaptic NMDA receptors and influence synaptic inhibition of Purkinje neurons. Journal of Neuroscience 24: 5659–5669. Iino M, Goto K, Kakegawa W, et al. (2001) Glia–synapse interaction through Ca2þ-permeable AMPA receptors in Bergmann glia. Science 292: 926–929.

Kirischuk S, Kettenmann H, and Verkhratsky A (1997) Naþ/Ca2þ exchanger modulates kainate-triggered Ca2þ signaling in Bergmann glial cells in situ. FASEB Journal 11: 566–572. Kirischuk S, Kirchhoff F, Matyash V, Kettenmann H, and Verkhratsky A (1999) Glutamate-triggered calcium signalling in mouse Bergmann glial cells in situ: Role of inositol1,4,5-trisphosphate-mediated intracellular calcium release. Neuroscience 92: 1051–1059. Kirischuk S, Moller T, Voitenko N, Kettenmann H, and Verkhratsky A (1995) ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells. Journal of Neuroscience 15: 7861–7871. Kirischuk S, Tuschick S, Verkhratsky A, and Kettenmann H (1996) Calcium signalling in mouse Bergmann glial cells mediated by a1-adrenoreceptors and H1 histamine receptors. European Journal of Neuroscience 8: 1198–1208. Kulik A, Haentzsch A, Luckermann M, Reichelt W, and Ballanyi K (1999) Neuron–glia signaling via alpha1 adrenoceptor-mediated Ca2þ release in Bergmann glial cells in situ. Journal of Neuroscience 19: 8401–8408. Matsui K, Jahr CE, and Rubio ME (2005) High-concentration rapid transients of glutamate mediate neural–glial communication via ectopic release. Journal of Neuroscience 25: 7538–7547. Muller T, Moller T, Berger T, Schnitzer J, and Kettenmann H (1992) Calcium entry through kainate receptors and resulting potassium-channel blockade in Bergmann glial cells. Science 256: 1563–1566. Muller T, Moller T, Neuhaus J, and Kettenmann H (1996) Electrical coupling among Bergmann glial cells and its modulation by glutamate receptor activation. Glia 17: 274–284. Rakic P (1971) Neuron–glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electron microscopic study in Macacus Rhesus. Journal of Comparative Neurology 141: 283–312. Reichenbach A and Wolburg H (2005) Astrocytes and ependymal glia. In: Kettenmann H and Ranson BR (eds.) Neuroglia, pp. 19–35. Oxford: Oxford University Press. Riquelme R, Miralles CP, and De Blas AL (2002) Bergmann glia GABAA receptors concentrate on the glial processes that wrap inhibitory synapses. Journal of Neuroscience 22: 10720–10730. Verkhratsky A, Orkand RK, and Kettenmann H (1998) Glial calcium: Homeostasis and signaling function. Physiological Reviews 78: 99–141. Warr O, Takahashi M, and Attwell D (1999) Modulation of extracellular glutamate concentration in rat brain slices by cysteine–glutamate exchange. Journal of Physiology 514: 783–793.