1 Experimental Neurobiology, Department of Neurosurgery, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
2 Bogomoletz Institute of Physiology, Bogomoletz St. 4, 01024 Kiev, Ukraine
3 Cellular Neurosciences, Max Delbrück Center for Molecular Medicine, Berlin, Robert Rössle Str., Germany
* Author for correspondence (e-mail: ronald.jabs{at}ukb.uni-bonn.de)
Accepted 26 May 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: GABAA receptor, GFAP, Glia, Glutamate, Hippocampus, Neuron-glia interaction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using transgenic mice expressing green fluorescent protein under control of the human GFAP promoter (hGFAP/EGFP mice), we have recently reported a co-existence of two types of glial cells in the hippocampus, distinguishable from each other by mutually exclusive expression of glutamate transporters (GluT type) and ionotropic glutamate receptors (GluR cells). GluT type cells were extensively coupled via gap junctions and contacted blood vessels, thus matching properties of classical astrocytes. By contrast, GluR cells lacked junctional coupling and did not enwrap capillaries (Matthias et al., 2003; Wallraff et al., 2004
). Moreover, GluR cells co-expressed S100ß, a common astrocyte marker, NG2, as well as neuronal genes, and hence escaped classification into neurons, astrocytes, or oligodendrocytes.
Here we used the hGFAP/EGFP transgenic animal to identify distinct types of glial cells in live slices. We combined ultrastructural analysis and post-recording immunocytochemistry to test whether the two populations of hGFAP/EGFP-positive glial cells in the hippocampus receive synaptic input. Electron microscopic inspection identified synapse-like structures with EGFP-positive postsynaptic compartments. Patch clamp recordings revealed stimulus-correlated as well as spontaneous postsynaptic events in GluR cells, but not in GluT cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vibratome sections (30-40 µm thickness) were cut perpendicularly to the longitudinal axis of the hippocampus on a Vibracut (FTB Feinwerktechnik, Bensheim, Germany). After permeabilization with 0.1% Triton X-100 and inactivation of endogenous peroxidase (latter step only for slices to be processed with HRP-coupled secondary antibodies), vibratome sections were incubated for 48 hours at 4°C with anti-GFP antibodies (rabbit IgG fraction, Molecular Probes, MoBitec, Göttingen, Germany) diluted 1:500 in blocking solution [5% BSA, 5% normal goat serum (NGS) in 0.1 M PB]. As controls, primary antibodies were omitted and slices were incubated in blocking solution. After extensive rinsing in PB, slices were incubated either with peroxidase-conjugated goat anti-rabbit IgG (1:200) (Dianova, Hamburg, Germany) or with goat anti-rabbit IgG conjugated to 1.4 nm gold (Nanogold; Nanoprobes, Yaphank, NY, USA) (1:40) for overnight at room temperature. After rinsing, slices incubated with HRP-conjugated secondary antibodies were developed using the standard diaminobenzidine (DAB) reaction. Slices probed with nanogold-coupled secondary antibodies underwent silver-intensified pre-embedding immunogold reaction as described (Baude et al., 1993). Subsequently, slices were post-fixed in osmium tetroxide, dehydrated in increasing series of ethanol, pre-embedded with propylene oxide and flat embedded in epoxy resin (agar 100 resin, araldite CY 212, DDSA, DMP-30; Plano, Wetzlar, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips 400 electron microscope at 80 kV or a JEOL 100CX electron microscope at 60 kV.
Post-embedding immunocytochemistry
hGFAP/EGFP transgenic mice (n=6) were perfused intra-cardially as described above with the following fixative: 4% paraformaldehyde, 0.05% glutaraldehyde and 0.2% of picric acid in 0.1 M PB (pH 7.4). After 15 minutes perfusion, the brains were removed, 500 µm thick vibratome sections were cut and sections were washed several times in PB. Freeze substitution and low temperature embedding in acrylic resins were carried out as described earlier (Baude et al., 1995; Nusser et al., 1997
). For cryoprotection, slices were placed into sucrose solutions (concentration 0.5-2.0 M) in 0.05 M Tris-maleat buffer. They were then slammed onto copper blocks cooled in liquid N2, followed by freeze-substitution with methanol and embedding in Lowicryl HM 20 (Chemische Werke Lowi GMBH, Germany) resins.
Lowicryl resin-embedded ultrathin sections (75-90 nm thickness) were picked up on formvar-coated copper grids and were incubated on drops of blocking solution consisting of TBS (50 mM Tris-HCl, pH 7.4, 0.3% NaCl) and 10% of NGS. Primary, anti-GFP antibodies (rabbit IgG, MoBiTec) were diluted 1:50 in TBS containing 2% NGS and sections were incubated on drops of antibody solution overnight at 4°C. Subsequently, sections were washed and incubated for 40 minutes with secondary antibodies (goat anti-rabbit IgG coupled to 12 nm colloidal gold; 1:100; Immunotech, Dianova). After several washing steps in PB and in ultra-pure water, the sections were contrasted with saturated aqueous uranyl acetate followed by staining with lead citrate. As a control, primary antibodies were either omitted and slices were incubated in blocking solution or replaced by 5% normal rabbit serum.
Slice preparation for electrophysiology and immunohistochemistry
Transgenic hGFAP/EGFP mice aged p9-p12 were anaesthetized, decapitated, the brains were removed. Hippocampal slices (300 µm) were cut perpendicularly to the main hippocampal axis using ice-cold oxygenated solution consisting of (in mM): 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, 25 glucose, 75 sucrose (347 mOsmol). The slices were stored for 30 minutes in the same solution at 35°C and then transferred into artificial cerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 3 KCl, 2 MgSO4, 2 CaCl2, 10 glucose 1.25 NaH2PO4, 26 NaHCO3, equilibrated with 95% O2 and 5% CO2 to a pH of 7.4 at room temperature.
Patch-clamp recordings
Slices were transferred to a recording chamber, and were constantly perfused with aCSF at room temperature. Whole-cell recordings were obtained using an EPC8 amplifier (HEKA Elektronik, Lambrecht, Germany). The holding potential in the voltage clamp mode was 80 mV, if not stated otherwise. In the current clamp mode, voltage signals were additionally amplified with a DPA 2F amplifier (NPI electronic GmbH, Tamm, Germany). Signals were digitized with an ITC 16 (NPI electronic). Patch pipettes, fabricated from borosilicate capillaries (Hilgenberg, Malsfeld, Germany), had resistances of 3-6 M when filled with a solution consisting of (in mM): 130 KCl, 2 MgCl2, 0.5 CaCl2, 3 Na2-ATP, 5 BAPTA, 10 HEPES. Experiments displayed in Figs 6, 7, 8 were performed with 125 K-gluconate, 20 KCl, 3 NaCl, 2 Na2-ATP, 2 MgCl2, 0.5 EGTA, 10 HEPES. The pH was adjusted to 7.25 for both internal solutions. Voltages were corrected for liquid junction potential (6 mV for the K-gluconate solution). Recordings were monitored with TIDA software (HEKA). Series and membrane resistance were checked in constant intervals with self-customized macros using Igor Pro 5.03 software (WaveMetrix Inc., Lake Oswedo, USA). Visual control was achieved with a microscope equipped with an infrared DIC system (Axioskop FS2, Zeiss, Oberkochen, Germany) and a 60x LUMPlan FI/IR objective (Olympus Optical Co., Hamburg, Germany). The infrared image was captured with an analogue tube camera and contrast enhanced with a controller (C2400-07, Hamamatsu Photonics, Herrsching am Ammersee, Germany).
|
|
|
Offline compensation of capacitative artefact
Membrane currents were offline compensated for stimulus artefacts using Igor Pro 5.03 software. Ten traces evoked by 10 mV voltage steps from 80 to 70 mV were averaged and fitted monoexponentially. Compensated current traces were obtained by multiplying the fitted curve by the respective factors and subsequent subtraction from the original current traces at different membrane potentials. Evoked glial PSCs (ePSCs) were compensated for stimulus artefacts by subtracting averaged failure traces.
Cell identification and immunohistochemistry
In this study, weakly fluorescent glial cells of the GluR type have been investigated, the properties of which have been reported in detail elsewhere (Matthias et al., 2003; Wallraff et al., 2004
). For morphological and immunohistochemical analysis, the recorded cells were labelled by adding a red fluorescent dye (0.1% dextran-conjugated Texas Red or 0.1% dextran-conjugated TRITC, MW 3000, Molecular Probes, Leiden, Netherlands) to the pipette solution. Slices were fixed overnight with 8% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) at 4°C. NG2 and S100ß immunoreactivity was tested through double labelling using immunofluorescent antibodies. After washing in Triton X-100 containing PBS, the tissue was incubated for 24 hours at 4°C with either a polyclonal rabbit antibody directed against S100ß (Swant, Bellinzona, Switzerland; 1:500), or a polyclonal rabbit antibody directed against NG2 (gift of W.P. Stallcup; 1:500). After repeated rinsing in PBS, the slices were further incubated with goat anti-rabbit immunoglobulins coupled to biotin (Dianova; 1:200) and visualized with the streptavidin-conjugated fluorochrome, indocarbocyanine (Cy5, Dianova; 1:200). The sections were mounted on slides using immunofluore mounting medium (Confocal-Matrix, micro-tech-lab, Graz, Austria) and evaluated using a confocal laser-scanning microscope in an inverted configuration (Leica TCS 4D, Leica, Bensheim, Germany). To avoid crossover of fluorescence between channels, sequential scanning was used with tight filter bands centred on the peak emissions of EGFP, TRITC and CY5. Cell morphology was visualized by taking consecutive optical sections up to a depth of 40 µm. Each channel was projected into a 2D micrograph using maximum intensity projection. The three greyscale pictures were combined by assigning them to the pseudo red (CY5), blue (EGFP), and green (Texas Red or TRITC) channels of an RGB picture. Immunoreactivity and the size of the cells were estimated using Metaview 4.5 software (Universal Imaging Corp., Downingtown, PA, USA). Therefore, background corrected 8 bit greyscale values of the tracer-filled cells were averaged and compared with the fluorescence intensity of adjacent immunopositive cells. The size covered by cell processes was measured as area. Reagents were purchased from Sigma unless otherwise stated. Data are given as mean ±s.d. Differences were tested for significance using the Student's t-test (P<0.05).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Sub-threshold stimulation of Schaffer collaterals and simultaneous current-clamp recording revealed synaptic innervation of GluR-type glial cells
To test whether GluR cells receive synaptic input, stimulation pulses were applied through a bipolar platinum wire electrode located in the Schaffer collaterals, and a glial cell was analysed with the patch-clamp method. Simultaneously, field potentials were monitored in the stratum radiatum with an electrode placed in the close vicinity (20-40 µm) of the recorded cell. Since evoked field potentials are not spatially homogenous over large areas, this approach allowed a controlled fine-tuning of the excitation level evoked in the tissue surrounding the analysed glial cell. To avoid the generation of postsynaptic action potentials and recurrent neuronal circuits, stimulation intensity was adjusted sub-threshold (50-150 microseconds, 50-250 pA, 120 stimuli at 1 second intervals). The presence of afferent fiber volleys and dendritic field potentials verified successful fibre tract stimulation and presynaptic transmitter release (Fig. 3B1). Under these conditions, current clamp recordings revealed stimulus-correlated depolarizations of the glial cell membrane (n=12/19 cells) (Fig. 3A1). Repetitive stimulation at constant intensity evoked postsynaptic potentials in GluR cells (ePSPs) of up to 8 mV, but also disclosed a significant failure rate (Fig. 3A2,C,D1). Analysis of amplitude histograms revealed a Gaussian distribution of baseline fluctuation of the glial membrane potential (Fig. 3D2), but the glial ePSPs were clearly non-Gaussian distributed (Fig. 3D1). This observation suggested quantal transmitter release at a neuron-glia synapse-like structure and predicted the existence of spontaneous glial PSPs. Indeed, low-frequency, spontaneous PSPs (sPSPs) (Fig. 3A3, bottom) occurred in most GluR cells tested (20/24).
|
|
Pharmacological analysis indicated monosynaptic GABAergic input onto GluR cells
Next we set out to identify the mechanism(s) underlying the depolarisation of GluR cells. Voltage clamp recordings unravelled fast- and slow-decay time constants of the evoked responses, and both components were generated by individual GluR cells (n=14). Typical examples of fast and slow glial ePSCs are given in Fig. 4A,B. Both components were activated within about 1 millisecond in a stimulus-correlated manner and displayed decay time constants of 1-5 milliseconds and about 20 milliseconds, respectively. Occasionally, we also noted responses with two decay time constants (Fig. 4C). As expected for synaptic events, spontaneous glial PSCs (sPSCs) were also observed. Individual GluR cells displayed fast, slow, or biphasic sPSC kinetics; see below (Fig. 8A2) (Table 1).
|
The identity of evoked (120 single stimulation pulses, interstimulus interval 3 seconds or 10 seconds) and spontaneous PSCs in GluR cells was investigated in the presence of antagonists of ionotropic glutamate and GABAA receptors. Application of NBQX (10 µM) completely abolished dendritic field potentials while the presynaptic fiber volley was still visible (Fig. 5A top panel). In the presence of this antagonist, slowly decaying glial ePSCs remained largely unchanged (n=3) (Fig. 5A lower panel). This indicated that (1) GluR cells received monosynaptic input and (2) a significant proportion of glial ePSCs were caused by receptors other than AMPA/kainate receptors. Application of bicuculline (10 µM) significantly and reversibly blocked the slow glial responses (n=4; Fig. 5B) while field potentials and fast, bicuculline-insensitive GluR cell ePSCs remained under these conditions (see below). These findings demonstrated that the slow responses were mediated by postsynaptic glial GABAA receptors.
|
To investigate further the properties of GABAA receptor mediated glial ePSCs, interneurons were activated more directly through near field stimulation, and the Cl concentration of the patch pipette solution was reduced to 27 mM to mimic physiological conditions. In the presence of NBQX (10 µM) and APV (25 µM D-APV or 50 µM DL-APV), ePSCs of GluR cells displayed a mean amplitude of 4.7±1.5 pA and decayed with a time constant of 25.4±7.2 milliseconds (80 mV, n=7). The failure rate was 69±15% (n=7) (Fig. 6B). The GABA mediated ePSCs could be completely blocked by 1 µM TTX (not shown). Reversal potential analysis was performed to confirm that the evoked glial responses were not due to GABA uptake. As a comparison, we first studied CA1 interneurons. Under our experimental conditions, GABAA receptor mediated spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 neurons in the whole cell configuration reversed at 40 mV, which was close to the theoretical Cl equilibrium potential (ECl=39.7 mV). Accordingly, changing the driving force for Clin GluR cells by shifting the holding potential from 80 to 0 mV, led to outwardly directed glial ePSCs (Fig. 6C). Amplitudes amounted to 5.1±2.4 pA (n=4), which did not differ significantly from the absolute value measured at 80 mV (cf. above). These findings corroborated the view that the glial ePSCs were due to Cl passing through GABAA receptors.
In addition, sPSCs were registered in GluR cells in the presence of NBQX and APV (9/10 cells; Fig. 6D-G), which did not differ in amplitude (5.0±1.9 pA, n=9) and decay time (20±12 milliseconds, n=9) from the ePSCs described above. In contrast to neuronal sIPSCs (Banks et al., 2002; Mody and Pearce, 2004
), the glial GABA mediated sPSCs occurred at very low frequencies (1.0±0.5 events per minute, n=9; Fig. 6D). To verify that these rare sPSCs did not represent irregular noise, sPSCs were provoked through depolarisation of presynaptic terminals by raising [K+]out. As expected, bath application of 10 mM KCl in the presence of NBQX and APV reversibly increased the incidence of GABA-induced sPSCs (to 32±16 events per minute; amplitude, 3.5±0.7 pA; decay time constant, 18.3±0.9 milliseconds, n=2) (Fig. 6F). Together, these data confirmed that the slow glial PSCs were due to activation of GABAA receptors in the GluR cell membrane.
Quantal analysis suggests a small number of presynaptic release sites giving rise to GABA mediated ePSCs in GluR-type glial cells
The GABAA receptor mediated glial sPSCs occurred at very low frequencies, and the evoked responses were characterized by small amplitudes and high failure rates. These properties indicated a low grade of synaptic innervation compared with hippocampal neurons. To achieve more information about the quantal nature of presynaptic GABA release onto the postsynaptic GluR cell membrane, the amplitude distribution of GABAA receptor mediated ePSCs was studied through independent (i.e. interstimulus interval 10 seconds) single pulse stimulation (Fig. 7). ePSCs averaged out at 5 pA, with a rise time of 1.8 milliseconds (Fig. 7A). Individual amplitudes of a given cell varied between 0 and 18 pA (e.g. Fig. 7B,C, n=4). Since the putative unitary amplitude and current noise were expected to be of the same order of magnitude, amplitudes for each cell were determined by averaging individual ePSCs within a fixed time window of 1 millisecond. The windows were placed at ±0.5 milliseconds of the averaged peak time for each cell. This allowed estimation of the noise-related error, which was characterised by the width of the Gaussian distribution around 0 pA (Fig. 7B,D,E). In contrast to baseline noise (Fig. 7D, inset), the ePSC amplitude histograms displayed non-Gaussian, binomial-like distributions, indicative of small numbers of release sites producing GABA-mediated glial ePSCs (Fig. 7D). Rough estimation yielded unitary amplitudes of about 1.3 pA. However, the superposition of amplitude histograms (n=4) did not display clear multiples of an unitary amplitude (Fig. 7E), even though all individual cells showed failures and ePSCs of non-Gaussian distributed amplitudes. Possible reasons for this finding will be discussed later on.
The rapid GABA-independent responses in GluR cells were inhibited by NBQX
As mentioned above, a second type of GluR cell PSCs were characterized by fast decay time constants (lower trace in Fig. 8A2). The rapid sPSCs and ePSCs were further analysed in the presence of the GABAA receptor antagonist, bicuculline (10 µM), which completely blocked the slow component. Bicuculline-resistant sPSCs occurred in all GluR cells tested (n=4; 3.1±3.0 events per minute) (Fig. 8A1,A3,A4). They displayed amplitudes of 6.0±2.1 pA, decayed with a time constant of 2.7±0.7 milliseconds, and were blocked by co-application of bicuculline with D-APV (25 µM) and NBQX (10 µM; n=3) (Fig. 8A5).
In the presence of bicuculline, short (100-150 microseconds) stimulation pulses also evoked rapid inward currents in all GluR cells tested (n=6) (Fig. 8B1). The bicuculline-resistant glial ePSC amplitudes (3.8±1.2 pA) and decay time constants (1.9±0.4 milliseconds) did not differ from the spontaneous responses. The failure rate was 65±21%. Co-application of bicuculline with NBQX and D-APV abolished the responses (n=5) (Fig. 8B2). This data suggested that in addition to GABA-mediated responses, PSCs were also produced by presynaptic release of glutamate that activated AMPA receptors in the postsynaptic GluR cell membrane.
Analysis of spontaneous PSCs under unblocked conditions identifies GABA and glutamate as the predominating neurotransmitters at neuron-to-GluR cell synapses
The results presented so far demonstrated that the majority of GluR cells displayed postsynaptic currents (50/57 cells) and revealed the existence of two independent types of synaptic input onto the glial cells. To determine whether the glial cells receive synaptic input other than glutamatergic or GABAergic, control sPSCs (i.e. in the absence of receptor antagonists) were investigated in more detail. Therefore, sPSCs of 20 individual GluR cells were sorted out according to their decay kinetics, and the fast and slow components were compared with properties of the pharmacologically isolated AMPA/kainate and GABAA receptor-mediated sPSCs, respectively. Frequency of occurrence, amplitudes and decay time constants of the control events did not differ from those of the separated glutamate- and GABA-mediated sPSCs reported above (Table 1). Thus, it is rather unlikely that additional neurotransmitters substantially contributed to synaptic innervation of GluR cells.
Both types of sPSCs coexisted in the majority of cells tested (14/20). Two cells showed only GABA- and four cells only glutamtate-mediated sPSCs. Together, these results show that most GluR cells in the CA1 stratum radiatum are innervated by both GABAergic and glutamatergic neurons, with the latter producing sPSC at a higher frequency.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Innervated GluR cells share properties of grey matter NG2 glia
To ascertain whether cells of the GluR type receive synaptic input, we recorded from cells in the hippocampal CA1 stratum radiatum displaying weak hGFAP-promoter activity as assessed by their low EGFP fluorescence intensity. Immunocytochemical and morphological analyses were performed subsequent to functional characterisation. All GluR cells tested expressed NG2, while about 30% of them were S100ß-positive. Despite the reduced recording time, it can not be excluded that washout of the relatively small S100ß molecule led to an underestimation of its expression. We noted that GluT cells, which represent `classical' astrocytes in the hippocampus often displayed higher S100ß immunoreactivity than GluR cells. The innervated GluR cells showed characteristic nodules, which occurred periodically along their processes. A similar morphology was described for so-called synantocytes, which are also NG2-positive (Berry et al., 2002).
For a long time NG2 was considered a marker of oligodendroglial precursor cells OPCs (Ong and Levine, 1999), but recent findings challenged this assumption (Butt, 2005
). Ultrastructural (Nishiyama et al., 2002
), immunohistochemical (Mallon et al., 2002
; Matthias et al., 2003
), and functional analyses (Chittajallu et al., 2004
) in the cortex or hippocampus indicated that NG2 glia comprise a heterogeneous cell population. Lineage analysis suggested that a subpopulation of postnatal NG2-positive cells in the early postnatal hippocampus represent neuronal progenitor cells (Belachew et al., 2003
) while the coexpression of GFAP or vimentin with NG2 after lesion to the adult brain (Alonso, 2005
) rather hints at an astroglial relationship of NG2 glia. Certainly, the cellular identity of these cells needs further consideration (Lin and Bergles, 2004
).
Are all GluR cells innervated?
The infrequent incidence and the small amplitudes required long recording periods and low background noise to pinpoint sPSCs in the glial cells, and the success rate of ePSC activation additionally depended on appropriate positioning of the stimulation electrode. Considering these conditions, our data indicated that a majority of the GluR cells received both, GABA- and glutamate-mediated synaptic input. Whether the small group of cells apparently lacking PSCs represented a separate subpopulation of GluR cells or rather reflected inadequate recording conditions is difficult to decide. We noted that the input resistance of innervated GluR cells was variable, ranging between 80 to 1.500 M (cf. Fig. S2 in supplementary material). However, it appeared that cells with lower values (Ri<20 M
) lacked both sPSCs and ePSCs. Further experiments are necessary to determine whether these cells comprise a distinctive subgroup of GluR cells devoid of synaptic input.
Are other neurotransmitters involved?
Expression of AMPA and GABAA receptors in GluR cells is well established (Jabs et al., 1994; Bekar et al., 1999
; Zhou and Kimelberg, 2001
; Matthias et al., 2003
). Our data indicate that GluR cells receive only glutamatergic and GABAergic input: (1) The frequency of spontaneous events in control conditions matched the sum of activity either under blocked glutamate or GABAA receptor-mediated transmission; (2) sPSCs are completely absent when ionotropic GABA and glutamate receptors are blocked (Fig. 8A5). Similar types of neuron-to-glia synapses were also described for NG2-positive presumed OPCs (Bergles et al., 2000
; Lin and Bergles, 2003
). We cannot completely exclude that part of the fast decaying sPSCs in GluR cells was caused poly-synaptically, by the release of transmitter(s) other than GABA or glutamate. Indeed, astrocytes in the hippocampus were activated by neuronal release of noradrenaline or acetylcholine, which, however, stimulated metabotropic receptors in the glial membrane (reviewed by Fellin and Carmignoto, 2004
; Volterra and Steinhäuser, 2004
). Nicotinic acetylcholine receptors and glycine receptors have been found in glial subpopulations in situ or in the cell culture, but not in acute hippocampal slices (reviewed by Kettenmann and Steinhäuser, 2005
). In conclusion, evidence available so far suggests that neuronal synaptic input onto GluR cells in the hippocampus activates postsynaptic AMPA and GABAA receptors only.
Properties of the neuron-glia signalling
A majority of the GluR cells received monosynaptic input from GABAergic neurons because in the case of polysynaptic circuits, block of glutamatergic transmission should have reduced sPSC frequency, which was not observed. Several reasons might account for the finding that quantal analysis of GABA-mediated glial ePSCs failed to reveal clear multiples of a unitary amplitude. First, the putative magnitude of the unitary current was in the range of 1 pA and therefore hardly to discriminate against current noise. However, reduction of noise due to superposition of histograms did not improve signal-to-noise relation. Therefore, a second explanation might be more appropriate. It is possible that GABA-mediated glial ePSCs have a varying quantal size, similar to neurons, where even single release sites show substantial variability (Ropert et al., 1990; Nusser et al., 2001
). In the case of cultured hippocampal neurons, the quantal size variation of GABAergic synapses is mainly caused by the speed of GABA clearance from the synaptic cleft, while fluctuations of the initial peak concentration play only a minor role (Barberis et al., 2004
). By contrast, ePSC kinetics remained unchanged in NG2-positive presumed OPCs after blockade of GABA transporters (Lin and Bergles, 2003
). The reason for the smearing of the amplitude histograms of GluR cell ePSCs as observed here, still has to be elucidated.
Another question concerns the biphasic decay of the glial ePSCs (Fig. 4C), which might result from co-activation of postsynaptic AMPA and GABAA receptors, or represent an intrinsic property of the GABA-induced responses. Although a contribution of AMPA receptors cannot be completely excluded, the latter assumption appears more likely because biphasic PSCs also occurred spontaneously (n=3, not shown), a situation where the probability of simultaneous release of GABA and glutamate from two different fibers should be rather low. Moreover, biphasic ePSCs were also observed after inhibition of AMPA receptors. Whether the biphasic desensitisation kinetics hinted at prolonged concentrations of high GABA concentrations in the neuron-glia cleft, as observed for GABAergic neuronal communication (Celentano and Wong, 1994; Mozrzymas et al., 2003
), remains to be investigated. Certainly, resolving the details of release mechanism and transmitter dynamics at GABAergic neuron-to-glia synapses represents a challenging task for future work.
Physiological role of neuron-to-GluR cell signalling
Astrocytes of the GluT type were shown to be in a position to release glutamate upon stimulation via regulated exocytosis (Bezzi et al., 2004), and to modulate the discharging pattern of neighbouring hippocampal neurons in situ (Fiacco and McCarthy, 2004
; Liu et al., 2004
; Angulo et al., 2004
; Fellin et al., 2004
, Volterra and Steinhäuser, 2004
). However, our data do not suggest that GluT cells receive direct synaptic input. Whether GluR cells are capable of releasing transmitters upon neuronal stimulation is still unknown. The finding that GluR cells generated network driven, spontaneous membrane depolarisations hinted at a physiological role of the neuron-to-glia signalling. Although the glial PSDs measured at the somatic membrane were rather small, receptor activation at distant processes of GluR cells might interfere with other transmembrane proteins, e.g. Kir channels (Schröder et al., 2002
) and lead to stronger, locally restricted depolarisations of subcellular microdomains, sufficient to activate intracellular signalling cascades. Future studies have to elucidate whether the neuronal input is important for NG2-mediated clustering of AMPA receptors in GluR cells (Stegmüller et al., 2003
) or is involved in more complex, e.g. growth factor-related glial reactions (Stallcup, 2002
).
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aguirre, A. A., Chittajallu, R., Belachew, S. and Gallo, V. (2004). NG2-expressing cells in the subventricular zone are type C-like cells and contribute to interneuron generation in the postnatal hippocampus. J. Cell Biol. 165, 575-589.
Alonso, G. (2005). NG2 proteoglycan-expressing cells of the adult rat brain: Possible involvement in the formation of glial scar astrocytes following stab wound. Glia 49, 318-338.[CrossRef][Medline]
Angulo, M. C., Kozlov, A. S., Charpak, S. and Audinat, E. (2004). Glutamate released from glial cells synchronizes neuronal activity in the hippocampus. J. Neurosci. 24, 6920-6927.
Banks, M. I., Hardie, J. B. and Pearce, R. A. (2002). Development of GABA(A) receptor-mediated inhibitory postsynaptic currents in hippocampus. J. Neurophysiol. 88, 3097-3107.
Barberis, A., Petrini, E. M. and Cherubini, E. (2004). Presynaptic source of quantal size variability at GABAergic synapses in rat hippocampal neurons in culture. Eur. J. Neurosci. 20, 1803-1810.[CrossRef][Medline]
Baude, A., Nusser, Z., Roberts, J. D., Mulvihill, E., McIlhinney, R. A. and Somogyi, P. (1993). The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron 11, 771-787.[CrossRef][Medline]
Baude, A., Nusser, Z., Molnár, E., McIlhinney, R. A. J. and Somogyi, P. (1995). High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus. Neuroscience 69, 1031-1055.[CrossRef][Medline]
Bekar, L. K., Jabs, R. and Walz, W. (1999). GABAA receptor agonists modulate K+ currents in adult hippocampal glial cells in situ. Glia 26, 129-138.[CrossRef][Medline]
Belachew, S., Chittajallu, R., Aguirre, A. A., Yuan, X., Kirby, M., Anderson, S. and Gallo, V. (2003). Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons. J. Cell Biol. 161, 169-186.
Bergles, D. E., Roberts, J. D., Somogyi, P. and Jahr, C. E. (2000). Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus. Nature 405, 187-191.[CrossRef][Medline]
Berry, M., Hubbard, P. and Butt, A. M. (2002). Cytology and lineage of NG2-positive glia. J. Neurocytol. 31, 457-467.[CrossRef][Medline]
Bezzi, P., Gundersen, V., Galbete, J. L., Seifert, G., Steinhäuser, C., Pilati, E. and Volterra, A. (2004). Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate. Nat. Neurosci. 7, 613-620.[CrossRef][Medline]
Butt, A. M. (2005). Structure and function of oligodendrocytes. In Neuroglia. 2nd edn (ed. H. Kettenmann and B. R. Ransom), pp. 36-47. Oxford: Oxford University Press.
Celentano, J. J. and Wong, R. K. (1994). Multiphasic desensitization of the GABAA receptor in outside-out patches. Biophys. J. 66, 1039-1050.[Abstract]
Chittajallu, R., Aguirre, A. and Gallo, V. (2004). NG2-positive cells in the mouse white and grey matter display distinct physiological properties. J. Physiol. 561, 109-122.
Fellin, T. and Carmignoto, G. (2004). Neurone-to-astrocyte signalling in the brain represents a distinct multifunctional unit. J. Physiol. 559, 3-15.
Fellin, T., Pascual, O., Gobbo, S., Pozzan, T., Haydon, P. G. and Carmignoto, G. (2004). Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43, 729-743.[CrossRef][Medline]
Fiacco, T. A. and McCarthy, K. D. (2004). Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons. J. Neurosci. 24, 722-732.
Greenwood, K. and Butt, A. M. (2003). Evidence that perinatal and adult NG2-glia are not conventional oligodendrocyte progenitors and do not depend on axons for their survival. Mol. Cell. Neurosci. 23, 544-558.[CrossRef][Medline]
Hama, K., Arii, T., Katayama, E., Marton, M. and Ellisman, M. H. (2004). Tri-dimensional morphometric analysis of astrocytic processes with high voltage electron microscopy of thick Golgi preparations. J. Neurocytol. 33, 277-285.[CrossRef][Medline]
Haydon, P. G. (2001). GLIA: listening and talking to the synapse. Nat. Rev. Neurosci. 2, 185-193.[CrossRef][Medline]
Jabs, R., Kirchhoff, F., Kettenmann, H. and Steinhäuser, C. (1994). Kainate activates Ca2+-permeable glutamate receptors and blocks voltage-gated K+ currents in glial cells of mouse hippocampal slices. Pflügers Arch. 426, 310-319.[CrossRef][Medline]
Kettenmann, H. and Steinhäuser, C. (2005). Receptors for neurotransmitters and hormones. In Neuroglia. 2nd edn. (ed. H. Kettenmann and B. R. Ransom), pp. 131-145. Oxford: University Press.
Kimelberg, H. K. (2004). The problem of astrocyte identity. Neurochem. Int. 45, 191-202.[CrossRef][Medline]
Lin, S. C. and Bergles, D. E. (2003). Synaptic signaling between GABAergic interneurons and oligodendrocyte precursor cells in the hippocampus. Nat. Neurosci. 7, 24-32.[CrossRef][Medline]
Lin, S. C. and Bergles, D. E. (2004). Synaptic signaling between neurons and glia. Glia 47, 290-298.[CrossRef][Medline]
Liu, Q. S., Xu, Q., Arcuino, G., Kang, J. and Nedergaard, M. (2004). Astrocyte-mediated activation of neuronal kainate receptors. Proc. Natl. Acad. Sci. USA 101, 3172-3177.
Mallon, B. S., Shick, H. E., Kidd, G. J. and Macklin, W. B. (2002). Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development. J. Neurosci. 22, 876-885.[CrossRef][Medline]
Matthias, K., Kirchhoff, F., Seifert, G., Hüttmann, K., Matyash, M. and Kettenmann, H. and Steinhäuser, C. (2003). Segregated expression of AMPA-type glutamate receptors and glutamate transporters defines distinct astrocyte populations in the mouse hippocampus. J. Neurosci. 23, 1750-1758.
Mody, I. and Pearce, R. A. (2004). Diversity of inhibitory neurotransmission through GABAA receptors. Trends Neurosci. 27, 569-575.[CrossRef][Medline]
Mozrzymas, J. W., Barberis, A., Mercik, K. and Zarnowska, E. D. (2003). Binding sites, singly bound states, and conformation coupling shape GABA-evoked currents. J. Neurophysiol. 89, 871-883.
Muller, A., Kukley, M., Stausberg, P., Beck, H., Müller, W. and Dietrich, D. (2005). Endogenous Ca2+ buffer concentration and Ca2+ microdomains in hippocampal neurons. J. Neurosci. 25, 558-565.
Newman, E. A. (2003). New roles for astrocytes: Regulation of synaptic transmission. Trends Neurosci. 26, 536-542.[CrossRef][Medline]
Nishiyama, A., Watanabe, M., Yang, Z. and Bu, J. (2002). Identity, distribution, and development of polydendrocytes: NG2-expressing glial cells. J. Neurocytol. 31, 437-455.[CrossRef][Medline]
Nolte, C., Matyash, M., Pivneva, T., Schipke, C. G., Ohlemeyer, C., Hanisch, U. K., Kirchhoff, F. and Kettenmann, H. (2001). GFAP promoter-controlled EGFP-expressing transgenic mice: A tool to visualize astrocytes and astrogliosis in living brain tissue. Glia 33, 72-86.[CrossRef][Medline]
Nusser, Z., Cull-Candy, S. and Farrant, M. (1997). Differences in synaptic GABAA receptor number underlie variation in GABA mini amplitude. Neuron 19, 697-709.[CrossRef][Medline]
Nusser, Z., Naylor, D. and Mody, I. (2001). Synapse-specific contribution of the variation of transmitter concentration to the decay of inhibitory postsynaptic currents. Biophys. J. 80, 1251-1261.
Ong, W. Y. and Levine, J. M. (1999). A light and electron microscopic study of NG2 chondroitin sulfate proteoglycan-positive oligodendrocyte precursor cells in the normal and kainate-lesioned rat hippocampus. Neuroscience 92, 83-95.[CrossRef][Medline]
Peters, A. (2004). A fourth type of neuroglial cell in the adult central nervous system. J. Neurocytol. 33, 345-357.[CrossRef][Medline]
Peters, A. and Palay, S. L. (1996). The morphology of synapses. J. Neurocytol. 25, 687-700.[Medline]
Pusch, M. and Neher, E. (1988). Rates of diffusional exchange between small cells and a measuring patch pipette. Pflügers Arch. 411, 204-211.[CrossRef][Medline]
Rietze, R., Poulin, P. and Weiss, S. (2000). Mitotically active cells that generate neurons and astrocytes are present in multiple regions of the adult mouse hippocampus. J. Comp. Neurol. 424, 397-408.[CrossRef][Medline]
Ropert, N., Miles, R. and Korn, H. (1990). Characteristics of miniature inhibitory postsynaptic currents in CA1 pyramidal neurones of rat hippocampus. J. Physiol. 428, 707-722.[Abstract]
Schipke, C. G. and Kettenmann, H. (2004). Astrocyte responses to neuronal activity. Glia 47, 226-232.[CrossRef][Medline]
Schröder, W., Seifert, G., Hüttmann, K., Hinterkeuser, S. and Steinhäuser, C. (2002). AMPA receptor-mediated modulation of inward rectifier K+ channels in astrocytes of mouse hippocampus. Mol. Cell. Neurosci. 19, 447-458.[CrossRef][Medline]
Stallcup, W. B. (2002). The NG2 proteoglycan: Past insights and future prospects. J. Neurocytol. 31, 423-435.[CrossRef][Medline]
Stegmüller, J., Werner, H., Nave, K. A. and Trotter, J. (2003). The proteoglycan NG2 is complexed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial progenitor cells. Implications for glial-neuronal signaling. J. Biol. Chem. 278, 3590-3598.
Steinhäuser, C., Jabs, R. and Kettenmann, H. (1994). Properties of GABA and glutamate responses in identified glial cells of the mouse hippocampal slice. Hippocampus 4, 19-36.[CrossRef][Medline]
Volterra, A. and Steinhäuser, C. (2004). Glial modulation of synaptic transmission in the hippocampus. Glia 47, 249-257.[CrossRef][Medline]
Wallraff, A., Odermatt, B., Willecke, K. and Steinhäuser, C. (2004). Distinct types of astroglial cells in the hippocampus differ in gap junction coupling. Glia 48, 36-43.[CrossRef][Medline]
Zhou, M. and Kimelberg, H. K. (2001). Freshly isolated hippocampal CA1 astrocytes comprise two populations differing in glutamate transporter and AMPA receptor expression. J. Neurosci. 21, 7901-7908.
|