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1Departments of Physiology and 2Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Canada
Submitted 18 August 2004; accepted in final form 20 October 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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70, 10, and <5% of total A current is associated with Kv4, Kv3, and Kv1 channels, respectively. In addition, pharmacology and kinetics provide evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Given this complex A-type channel expression pattern, we assessed the role of A currents in membrane voltage oscillations in response to current injections. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. As in neurons, this effect may enable astrocytes to respond rapidly to high-frequency synaptic events. Our results indicate that hippocampal astrocytes in vitro express multiple A-type Kv channel 
-subunits with accessory, possibly Ca2+-sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Function of these channels remains to be determined in a physiological setting. However, this study suggests that they enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). However, their role in glial cells, a nonexcitable cell type, is yet to be fully understood.
Astrocytes are the most numerous cells in the nervous system and appear to fulfill a wide range of neuronal support functions. These functions primarily involve regulation of external environment as well as trophic and metabolic support for active neurons ( Gard et al. 1995; Saad et al. 1991; Tsacopoulos 2002). As essential to these homeostatic functions, astrocytes express many ligand ( Porter and McCarthy 1997) and voltage-gated ( Bordey and Sontheimer 2000; Lascola et al. 1998; Latour et al. 2003) channels. Astrocytic voltage-gated potassium channels in particular can be subclassified as delayed rectifiers, rapidly inactivating A-type, and inwardly rectifying channels ( Sontheimer 1994; Sontheimer and Waxman 1993). Surprisingly, although many studies have looked at the pharmacological profile of voltage-gated potassium channels in astrocytes ( Bordey and Sontheimer 1999), little is known regarding functional channel subunit expression, of inactivating A-type channels in particular. Furthermore, although delayed rectifying Kv currents have been implicated in cell cycle processes ( Gallo et al. 1996; Ghiani et al. 1999; Knutson et al. 1997; Pappas et al. 1994), a physiological function for A-type channels in this nonexcitable cell have yet to be demonstrated.
Considering the absence of information regarding the molecular identity and function of inactivating A-type channels, the objective of the following study was to better characterize A-type channel expression in cultured astrocytes to aid in understanding the function of these rapidly inactivating potassium currents. Pharmacology demonstrates astrocytic A-currents to be predominantly composed of Kv4 subunits with KChIP accessory subunits. In addition, astrocytic A-type current kinetics most closely resemble Kv4 family kinetics. Furthermore, immunocytochemical and RT-PCR analysis of hippocampal astrocyte cultures confirm pharmacological and kinetic studies. Given the complex expression pattern for A-type currents in astrocytes, the role of A-currents in membrane voltage oscillations in response to current injections (simulating rapid glutamate-mediated synaptic-like events) was assessed to help provide insight into A-current function. We found that although TEA-sensitive delayed rectifying Kv currents are involved in the extent of repolarization possibly leading to afterhyperpolarizations (AHP), 4-aminopyridine (4-AP)-sensitive A-currents serve to increase the rate, enabling astrocytes to respond rapidly to high-frequency current injections.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Primary cultures of hippocampal astrocytes were prepared from 1-day-old Wistar rat pups. Animals were anesthetized with methoxyflurane (Janssen Pharmaceutica) and decapitated. Hippocampi were removed aseptically and cells were mechanically dissociated using an 80-µM Nitex filter. The cells were seeded (105 cells/35-mm dish) in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, Oakville) containing 20% low endotoxin (
10 EU/ml) defined donor horse sera (Hyclone) and 20 µg/ml gentamicin (Invitrogen) and incubated at 37°C in humidified 5% CO2-95% O2. After 3 days, the culture medium was replaced with DMEM containing 10% low endotoxin horse sera. Medium replacement was then repeated twice a week for the continuation of the culture process. Cells were used for whole cell patch recordings when cells formed a confluent monolayer (10–12 days).
Hek293 cell culture and transfection
Frozen HEK cells (ATCC) in 5% DMSO and 20% fetal bovine serum (FBS) containing DMEM were rapidly thawed at 37°C and plated out in 100-mm culture dishes. Media was changed every 2–3 days. Cells were passaged and replated prior to reaching confluence. The rat Kv1.4 clone ligated into SalI/BamHI double-digested site of the pBK-CMV vector and the rat Kv4.2 clone ligated into HindIII/NotI restriction sites of the pRC/CMV vector were kind gifts from Dr. J. Nerbonne. For transfections, HEK cells were plated in 10% FBS containing DMEM at 8x105 cells/60-mm dish 1 day prior to transfection. On the day of transfection, the media was replaced with 5 ml 10% FBS containing DMEM without antibiotics. Plasmid DNA containing cDNA of channel of interest (0.5 µg Kv1.4, 1.5 µg Kv4.2) and a GFP-containing plasmid (1.0 µg pIRES2-eGFP, Clontech) were diluted in a glass vial containing raw DMEM without antibiotics and serum (400 µl). In a separate glass vial, Lipofectamine 2000 (6–8 µl, Invitrogen) was also diluted (400 µl) and subsequently added to the vial containing the diluted cDNA within 5 min. The mixture (800 µl) was then allowed to sit for 30 min at room temperature for Lipofectamine-plasmid complexes to form before adding mixture dropwise to the 60-mm culture dish. Cultures were incubated for 4–24 h in the culture incubator before removing the media and adding fresh 10% FBS DMEM with antibiotics. Cells were allowed to grow 48 h after transfection at which time the cells were trypsinized (removed), counted and replated in 35-mm dishes at 3x105 cells/dish for subsequent patch experiments 2–10 h later.
RNA isolation and RT-PCR analysis
Total RNA was isolated from confluent monolayer cultures of astrocytes from rat brain by scraping cells in 1 ml of TRIZOL reagent (Invitrogen) containing guanidine thiocyanate and phenol. After addition of 0.2 ml chloroform, tubes were centrifuged at 12,000 g for 15 min at 4°C. RNA in the supernatant solution was precipitated by adding an equal volume of ice-cold isopropanol and pelleted by centrifugation at 12,000 g for 10 min at 4°C and washed by 75% ethanol. RNA pellets were resuspended in RNase-free water (Invitrogen). RT-PCR was performed using SuperScript One-Step RT-PCR with Platinum Taq from Invitrogen. Both reverse transcription and cDNA amplification were performed in a 50-µl reaction mixture containing 300 ng of total RNA, 25 µl of the proprietary 2x reaction buffer containing 0.4 mM of each dNTP and 2.4 mM MgSO4 and 1 µl RT/Platinum Taq mix containing SuperScript II Reverse Transcriptase and Taq DNA Polymerase. Sense and anti-sense primers (0.2 µM) were added and volume was brought to 50 µl with RNase-free water. The annealing temperature for PCR reaction cycles was adjusted according to the optimal annealing temperatures for each specific primer set. PCR products in a 6-µl aliquot were subjected to electrophoresis in 2.0% agarose gel in 1x TEA buffer and visualized with ethidium bromide. All primers were designed based on the specific region for selected Kv, 
, and KChIP subunits (Table 1).
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Double-labeling immunocytochemistry was performed using antibodies specific for Kv1.4, Kv3.4, or Kv4.3 in conjunction with the specific astrocytic marker (in culture) glial fibrillary acidic protein (GFAP). Cultures were fixed in 4% paraformaldehyde in phosphate-buffered saline for 30 min and blocked with antibody diluent at room temperature for 1 h. Next, the primary polyclonal rabbit anti-GFAP antibody (1:500; DAKO, Carpinteria, CA) was applied in combination with the monoclonal rabbit anti-Kv1.4 antibody (1:100; Upstate Biotechnology, Lake Placid, NY) and incubated overnight at 4°C. Alternately, the primary polyclonal rabbit anti-Kv3.4 or Kv4.3 antibodies (1:100; Alomone Labs, Jerusalem, Israel) were applied in combination with the polyclonal goat anti-GFAP (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and incubated overnight at 4°C. Goat anti-mouse TRITC (1:200 for Kv1.4) or donkey anti-rabbit FITC (1:200 for Kv3.4 and Kv4.3) were applied in conjunction with donkey anti-rabbit FITC (1:100) or donkey anti-goat TRITC (1:200) antibodies for 3 h at room temperature in the dark. Cultures were washed and coverslips mounted with Citiflour (Marivac Laboratories) to minimize quenching. Digital images were taken with a cool CCD Spot RT Color camera on an Olympus IX71 inverted fluorescence equipped microscope.
Electrophysiology
For electrophysiological recordings, 35-mm culture dishes were placed on an inverted microscope (Zeiss Axiovert 10—equipped with epifluorescence) and perfused by a gravity-fed 16-gauge needle. Perfusate was sucked off using a vacuum pump attached to an aspirator (Harvard Apparatus) that reduced fluctuations in volume. Extracellular perfusate contained (in mM) 126 NaCl, 24 NaGluconate, 3.5 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, and 5 HEPES and pH adjusted to 7.3 with NaOH. Borosilicate capillaries containing a thin filament (Harvard Apparatus), with a 1.5-mm OD and 0.33-mm wall thickness, were pulled to a tip diameter of <1 µm on a Flaming/Brown micropipette puller (Model P-97; Sutter Instruments) showing resistance of 2–4 M
when filled with (in mM) 130 KCl, 0.5 CaCl2, 2 MgCl2, 3 Na2ATP, 5 EGTA, and 10 HEPES and pH adjusted to 7.3 with KOH. The drugs tetraethylammonium (TEA), 4-AP, and 5,8,11,14-eicosatetraynoic acid (ETYA) were applied through the gravity-fed perfusion system. All chemicals were obtained from BDH (Toronto) or Sigma (St. Louis, MO) unless otherwise stated.
Patch-clamp recordings were obtained using the Axopatch-200B amplifier. Whole cell data were acquired with an acquisition frequency of 10 kHz, filtered at 2 kHz, and stored using a PC computer equipped with the Digidata 1200 digital/analog converter and PClamp 8.2.0.231 [EC] software (Axon Instruments).
Statistics and analysis
Steady-state activation and inactivation data were normalized and fit with a Boltzmann function. All inactivation time constants were obtained by fitting a single-exponential function to the decaying phase of currents. Both paired and unpaired t-test were used when appropriate to establish significance between groups. Results were deemed statistically significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Pharmacological studies were conducted on isolated inactivating A-type currents in astrocytes using the voltage subtraction protocol described in Fig. 1A. The isolated A-type current activated around –50 mV with peak currents averaging 1.96 ± 0.20 (mean ± SD) nA and showed nearly complete inactivation within the 500-ms voltage jumps (Fig. 1A). The impact of TEA on these isolated A-type currents was evaluated using the voltage subtraction protocol with a voltage jump from –80 to +60 mV repeated every 20 s. Perfusion with 10 mM TEA resulted in a 10 ± 2.5% steady-state block of A-current amplitude (Fig. 1B; n = 22). Although TEA is largely used as a blocker of delayed rectifying Kv channels, Kv3 (Shaw) family subunits display high sensitivity block to TEA making it, under the voltage subtraction protocol used, a selective antagonist for Kv3.3- or Kv3.4-inactivating A-type channels ( Fernandez et al. 2003; Riazanski et al. 2001). Subsequent pharmacological sensitivity to channel state-dependent 4-AP block was used to discriminate Kv4 family A currents from Kv1 as 4-AP binds to Kv4 channels in the closed state and Kv1 in the open state ( Tseng 1999). Voltage jumps from –80 to +60 mV were used as illustrated in Fig. 1C. The A-current amplitude immediately after 3 min of 4-AP perfusion was determined as closed-state block with the subsequent maximum block on additional voltage jumps representing open-state block. As this protocol was performed in the presence of 10 mM TEA, application of 4 mM 4-AP demonstrated an additional 70 ± 6.1% closed-state block and a 5.6 ± 3.6% open-state block, representing a total 4-AP block of 76 ± 4.3% (Fig. 1D; n = 9). These results are consistent with the majority of the TEA-insensitive A current being made up of Kv4 family subunits. The small open state block observed may suggest Kv1 family contribution consisting of Kv1.4 or other Kv1 subunits with Kv subunits. Additionally, the small open-state block may be a result of insufficient perfusion with 4-AP in this particular experimental design; this possibility was not further explored.
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50% in CHO cells ( Holmqvist et al. 2001) and Xenopus oocytes ( Villarroel and Schwarz 1996)]. In addition, KChIP modification of the rate of Kv4 channel inactivation is also disrupted by AA ( Holmqvist et al. 2001). ETYA was used because AA metabolites are known to have many downstream effects in astrocytes ( Ferroni et al. 2003; Harder et al. 1998). To avoid dilution of ETYA-mediated Kv4 effects with contaminating ETYA-insensitive Kv3.3/3.4 currents, these studies were conducted in the presence of 10 mM TEA, and the voltage subtraction method was utilized as in Fig. 1A. Application of 10 µM ETYA resulted in a 38 ± 3.9% block in amplitude from 1.32 ± 0.26 to 0.83 ± 0.20 nA (n = 7; Fig. 2), demonstrating again, a Kv4-family-specific effect in astrocytes. Interestingly, when normalized to control traces (Fig. 2A, inset), ETYA also showed a significant 37 ± 7.0% decrease in the time constant (tau) of inactivation from 31 ± 4.5 to 18 ± 1.9 ms (Fig. 2B), consistent with effects on Kv4/KChIP complexes ( Holmqvist et al. 2001).
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Astrocytic current patterns were compared with current patterns of Kv1.4 and Kv4.2 expressed in HEK293 cells to help clarify Kv subunits involved in the astrocytic A-type inactivating current pattern (Fig. 3). A-type inactivating outward K+ currents make up 73 ± 0.9% (n = 19) of total outward current in astrocytes as determined using a subtraction protocol based on voltage inactivation properties of A-type inactivating currents. The isolated A-type current kinetics were then compared with inactivating currents expressed in HEK cells (Fig. 3). Astrocytes and HEK cells expressing either Kv1.4 or Kv4.2 demonstrated sizeable rapidly inactivating current patterns (Fig. 3A). The rate of current inactivation was fit to single-exponential curves and inactivation rate constants (tau) were plotted at multiple voltages for comparison (Fig. 3A, inset). Astrocytic inactivation rates most closely resemble that of the rate of Kv4.2 inactivation. Kv1.4 inactivation rates were almost twice that of astrocytes. Although inactivation rates resemble those of Kv4.2 in HEK cells, astrocytic inactivation demonstrates a decrease in rate at more depolarized potentials. This departure from Kv4.2 kinetics may be due to additional A-type channel involvement but is also consistent with the possible involvement of additional cytoplasmic accessory subunits ( An et al. 2000; Holmqvist et al. 2002), such as that illustrated earlier in astrocytes by ETYA pharmacology.
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subunits. Steady-state inactivation kinetics show astrocytes (n = 21) to have a more hyperpolarized half inactivation point than both Kv1.4 (n = 5) and Kv4.2 (n = 9) in HEK cells (-67.79 ± 0.97, –54.99 ± 0.88, and –51.25 ± 1.38, respectively; Fig. 3B). However, the Kv4.2 slope width is the same as that of astrocytes (6.90 ± 0.60 vs. 6.58 ± 0.27), whereas Kv1.4 shows a significantly steeper slope (3.84 ± 0.54; Fig. 3B). Considering KChIP subunits had a dramatic 40-mV hyperpolarizing effect on Kv4.2 channel steady-state activation expressed in CHO cells ( An et al. 2000) and 9-mV hyperpolarizing shift on steady-state inactivation but not activation in HEK cells ( Van Hoorick et al. 2003), it is possible, once again, that the hyperpolarizing shift in astrocytic steady-state inactivation compared with Kv4.2 in HEK cells is a result of accessory subunit expression. Astrocytic activation kinetics demonstrate a slope width significantly longer than kinetics in HEK cells (20.17 ± 0.93, 9.67 ± 1.07, and 12.09 ± 0.66 for astrocytes, Kv1.4 and Kv4.2, respectively; Fig. 3C). However, astrocyte half activation (–1.83 ± 1.73; n = 37) shows a greater similarity to Kv4.2 (–1.27 ± 1.80; n = 10) than Kv1.4 (–38.85 ± 0.69; n = 16; Fig. 3C). Astrocytic reactivation demonstrates the greatest similarity to the Kv4 subfamily because of their rapid reactivation kinetics. Reactivation kinetics for cultured astrocytes shows a time constant of 10.15 ± 1.07 ms (n = 8; Fig. 3D) compared with a much longer 2,650 ms (n = 2) for Kv1.4 in HEK cells (data not shown). These reactivation kinetics are more rapid than findings by Van Hoorick et al. (2003) in HEK cells where Kv4.2 demonstrated recovery time constants of 242 ms alone or half that with KChIP1a or KChIP1b. Taken together, although there are definite differences with comparison to Kv4.2 alone, astrocytic kinetics appear consistent with inactivating currents being comprised of Kv4 subfamily members with cytoplasmic accessory subunits (Fig. 3). Immunocytochemistry and RT-RCR support astrocytic Kv channel pharmacology and kinetics
Due to pharmacologically elucidated expression of several A-type inactivating channel subunits and the interesting finding that A-current kinetics in astrocytes are more complicated than those of Kv4.2 expression alone in HEK cells, immunocytochemical and RT-PCR analysis was performed to provide additional insight into astrocytic A-current complexity. Glial fibrillary acidic protein-positive, stellate astrocytes in culture were probed using specific antibodies for a single member from each of the three Kv channel subfamilies. Expression patterns demonstrate site-specific targeting with Kv3.4 localized primarily to cell processes and Kv4.3 channels showing intense staining throughout both cell soma and processes (Fig. 4, A and B, respectively). The observation that Kv4.3 immunostaining is dominant in the cell soma is consistent with whole cell patch-clamp kinetics and pharmacology. Kv1.4 immunostaining could not be discriminated from background levels, suggesting minimal to no expression (data not shown). As a positive control however, prominent Kv1.4 staining could be obtained in transiently transfected HEK293 cells under identical conditions (data not shown).
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; Holmqvist et al. 2002; Morohashi et al. 2002; Takimoto et al. 2002). Screening for the two members Kv3.3 and Kv3.4 from the Shaw subfamily also proved successful (Fig. 5B), although with reduced intensity compared with Shal subfamily members and GAPDH) used as a general cell marker for comparison (Fig. 5B, right). Although immunocytochemical analysis did not provide evidence for the presence of Kv1.4, pharmacological data suggests 5% of the A currents may be due to Shaker-related channel subunits. Therefore expression of Kv1.4 from the Shaker family and the auxiliary -subunit possibilities Kv1.1–1.3, Kv2.1 and Kv3.1 were examined as combinations of these subunits with subunits may constitute rapidly inactivating channels ( Heinemann et al. 1996; Pongs et al. 1999). Interestingly, mRNA for Kv1.4 was found in these hippocampal cultures (Fig. 5C). In addition, the subunits Kv1.1, 2, and 3, but not Kv1.2 or 1.3 (not shown) were also found (Fig. 5C, right). The subunits 1.1 and 3.1 are able to confer rapid inactivation onto several of the Kv subunits ( Kwak et al. 1999; Rettig et al. 1994), whereas the Kv2.1 appears to primarily regulate expression of channels at the membrane surface ( Manganas and Trimmer 2000; Shi et al. 1996); although it has been shown to alter the activation and inactivation properties of the Kv1.4 channel subunit ( McCormack et al. 1995).
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Current injection studies show a distinct role for A currents in shaping the repolarizing phase of the membrane voltage waveform (Fig. 6) ( Bordey and Sontheimer 1999). Consistent with previous studies, whole cell current-clamp evaluation of 20-ms 400-pA current injections demonstrate astrocytes have the ability to spike (Fig. 6) ( Sontheimer et al. 1992; Bordey and Sontheimer 1998a, b, 1999). Pharmacological analysis demonstrates that TEA-sensitive K+ currents impact the AHP but not the width of the waveform (Fig. 6A), whereas 4-AP has a dramatic effect on the width (Fig. 6B). This effect can be seen more clearly in the same cells as Fig. 6, A and B, but with a very short current injection allowing the spike to repolarize back to cell resting potential completely on its own (Fig. 6, C and D, respectively). The slash mark on the depolarizing phase of Fig. 6, C and D, indicates the time point at which the membrane was clamped back to 0 current, allowing visualization of spiking behavior. Application of 10 mM TEA, although causing a slight increase in amplitude, does not affect the repolarizing phase back to resting potential (Fig. 6C). On the other hand, 4 mM 4-AP clearly prolongs the repolarization phase back to resting potential (Fig. 6D). It is interesting to note the tracings in Fig. 6, C and D, do not show AHPs compared with traces from the same cells in A and C, respectively. This is likely a result of the astrocyte resting membrane potential being highly negative close to the equilibrium potential of K+. In support of this possibility, cells with a slightly depolarized resting potential (–40 to –47 mV; n = 5) always displayed an AHP with similar current injection to that shown in Fig. 6, C and D (data not shown).
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200 Hz ( Klausberger et al. 2003; Kunec and Bose 2003; Ponomarenko et al. 2003). Therefore astrocytes under current-clamp control were subjected to current injections at 100 and 200 Hz before and after administration of either 10 mM TEA or 4 mM 4-AP to assess the roles of delayed and A-type current in membrane responses (Fig. 7). Aside from the small depolarizing effect of TEA on resting membrane potential (Fig. 7A), TEA did not appear to dramatically alter the astrocytes ability to respond to subsequent stimuli at high frequency as evident when the current traces are overlaid (Fig. 7A, bottom). In contrast, 4-AP had a dramatic effect on frequency oscillations (Fig. 7B). The same prolongation of the repolarization phase seen in Fig. 6B results in summation leading to a more rapid and sustained membrane depolarization without altering the amplitude of individual spikes (Fig. 7B, bottom).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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70, 10, and <5% of total A currents are associated with Kv4, Kv3, and Kv1 channels, respectively (Fig. 1). The remaining 15% may possibly be explained as an incomplete block by TEA and 4-AP. Furthermore, ETYA pharmacology and kinetic analysis provides evidence for a significant contribution of KChIP accessory proteins in the complexity of astrocytic A currents (Figs. 2 and 3, respectively). This breakdown of electrophysiological expression is consistent with the Kv4 family showing bright ubiquitous staining in immunocytochemistry experiments (Fig. 4B) and the densest bands in PCR (Fig. 5A). The small contribution to inactivating A currents by members of the Kv3 family is perhaps a result of targeted expression to astrocytic processes, where contribution to whole cell currents would be limited due to space clamp control issues (Fig. 4A). The targeted expression of specific A-channel subunits does, however, suggest that these channels serve a specific function. Consistent with this, current-clamp studies demonstrate that A currents increase repolarization, limiting the extent and duration of astrocyte membrane depolarizations in response to current injection. Does channel localization provide insight to function?
Although the function of A channels in astrocytes is yet to be determined, they appear to be targeted to specific subcellular membrane locations. The three different Kv channel families involved in creating A-type inactivating potassium currents differ in their kinetics and therefore function. Targeted localization in neurons of Kv4 subunits to soma and dendrites ( Shibasaki et al. 2004; Song et al. 1998), Kv3.4 subunits to the axosomatic compartment ( Riazanski et al. 2001; Veh et al. 1995) and Kv1 subunits to terminals ( Monaghan et al. 2001; Sheng et al. 1993) is a testament to this fact. Localization of Kv3.4 channels to astrocytic cell processes in this study suggest this is a mechanism independent of environment or activity and may be a property of the gene family itself. Furthermore, although these studies focus on astrocytes visualized in culture, current patterns are not inconsistent with current patterns in situ ( Bekar et al. 1999; Bordey and Sontheimer 1997, 2000) or acutely isolated astrocytes ( Schools et al. 2003; Zhou and Kimelberg 2000). Therefore perhaps the functional significance of targeting inactivating channels in neurons can be used to better understand the function behind the expression patterns found in astrocytes.
Although Kv1.4 immunostaining could not be demonstrated above background levels in cultured stellate astrocytes, PCR results demonstrate the presence of Kv1.4 mRNA. Kv1.4 mRNA in these heterogeneous cultures may be a result of small contaminating populations of microglia, endothelial cells and/or fibroblasts. Alternatively, it may be that the density is very low in astrocytes, and the channels may be targeted, as in neurons, to astrocytic endfeet terminals. Kv1.4 subunits may also be forming heteromultimers with Kv1.1 or 1.2 subunits ( Monaghan et al. 2001; Sheng et al. 1993) diluting immunopuncta, making positive staining above background very difficult. In any case, voltage-clamp studies demonstrate minimal Kv1 family contribution to astrocytic A currents (5%; Fig. 1D).
KChIPs: A-type Kv channel calcium sensors?
Given the implications for calcium waves and oscillations in astrocytes, it is not surprising to find evidence for astrocyte expression of potassium-channel-interacting proteins (KChIPs; Figs. 1 and 5) from the larger neuronal calcium sensor-"recoverin" gene subfamily ( An et al. 2000). KChIPs contain four EF-hand-like motifs ( An et al. 2000), suggesting they may provide Ca2+ sensitivity to the A-type Kv4 channel family. Indeed, frequenin (also known as neuronal calcium sensor 1; NCS-1) from the same gene subfamily has been shown to localize and modulate Kv4.2 currents in a Ca2+-dependent manner ( Nakamura et al. 2001) with increasing Ca2+ levels prolonging the rate and increasing recovery of Kv4 inactivation. KChIP modulations of astrocytic A current in this study suggest that part of the astrocytic A current may be Ca2+ sensitive. This may serve a negative feedback role to allow an increase in outward K+ flux for increased repolarization under high activity conditions. On a side note, KChIP sensitivity to physiological concentrations of AA in astrocytes provides an interesting link to synaptic plasticity events, as AA is thought to play a major role in LTP ( Chen et al. 2002; Ramakers and Storm 2002).
Endogenous hippocampal neuronal firing frequency is consistent with astrocytic signaling capability
The hippocampus is heavily involved in encoding, consolidation, and retrieval of information by means of frequency-dependent network oscillations. Theta oscillations (4–10 Hz) of hippocampal pyramidal cells and interneurons occur during exploratory behaviors and rapid-eye-movement (REM) sleep, whereas sharp-wave firing of CA3 pyramidal neurons is associated with high-frequency (120–200 Hz) ripples in CA1 dendritic fields during consummatory behaviors and slow-wave sleep ( Klausberger et al. 2003; Kunec and Bose 2003; Ponomarenko et al. 2003). In fact, burst firing may be required to induce synaptic modifications in hippocampal circuits as well as projections to neocortical targets that participate in memory consolidation ( Buzsaki et al. 1987). As astrocytic Ca2+ oscillations and syncytial Ca2+ waves are thought to play a role in long-term potentiation and modification of synaptic connections ( Dani et al. 1992; Liu et al. 2004; Nagai et al. 2004), it is interesting to note that they appear to be augmented under conditions of neuronal bursting behavior ( Hirase et al. 2004; Latour et al. 2001). In addition to high-frequency-mediated Ca2+ responses, it is known that astrocytes also display sizeable synaptically induced currents (100 Hz) ( Diamond and Jahr 2000), suggesting they may be significantly depolarized. Therefore it appears that hippocampal astrocytes are subjected to high-frequency stimulation that can result in significant depolarization, possibly creating a scenario for recruitment of inactivating Kv currents in membrane repolarization.
Perspectives and limitations
A major obstacle to ascribing a function to astrocytic A currents is providing a physiological scenario where the cell membrane is depolarized rapidly enough to activate them. Using current injections in this study provided an experimental situation to evaluate potential roles of these currents. However, it remains to be determined if this experiment simulates any endogenous circumstance. Given that many astrocyte preparations ( Bordey and Sontheimer 1998a, b, 1999; Labrakakis et al. 1997; Patt et al. 1996; Sontheimer et al. 1992) demonstrate a slow glial "spike" similar to that seen in this study, A currents may subsequently be activated in response to rapid NT depolarization (<3 ms) ( Matthias et al. 2003; Seifert and Steinhauser 1995) sufficient to elicit a glial spike that typically depolarizes cells up to –20 or 0 mV (Fig. 6; well above threshold for activation of A current). Such a situation may occur in the confines of the synaptic cleft. The major argument, however, opposing such a scenario is the fact that astrocytes have significant inwardly rectifying K+ currents making it difficult to elicit this spike in situ. The preparations mentioned earlier were of astrocytes in culture or associated with different pathological states in vivo. We propose that under high-frequency stimulation, with concomitant glutamate-mediated block of inward rectifying currents ( Schroder et al. 2002), a slow glial spike is unmasked, potentiating and accelerating NT-mediated membrane oscillations that enable astrocytic membrane responses to better mirror synaptic events. Support for this will undoubtedly need to come from studies in live animals or acutely isolated slice preparations where functional synapses remain intact. Dual electrophysiological recordings and/or multi-photon confocal imaging techniques now provide the resolution required to enable observation of astrocyte membrane responses to synaptic/axonal firing events with perhaps the additional ability to correlate astrocyte membrane responses with calcium signaling.
A currents in astrocytes may be important for synchronization with neuronal activity
Expression of multiple voltage gated channel currents in the so-called "nonexcitable" astrocyte may seem paradoxical at first but might provide astrocytes with the ability to establish rapid membrane oscillations in response to high K+ and neurotransmitter release from rapidly firing neurons. This ability to mirror neuronal activity may allow astrocytes to maintain membrane depolarization gradients across the glial syncytium for K+ buffering and siphoning capability.
In summary, our results indicate that hippocampal astrocytes in vitro express multiple A-type Kv channel subunits with accessory, possibly Ca2+-sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Furthermore, it is hypothesized that these channels, possibly in concert with glial voltage-gated Na+ channels, enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency signals, perhaps synchronizing glial functions with neuronal activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. Walz, Rm B39 Health Sciences Bldg, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada (E-mail: walz{at}sask.usask.ca)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Bekar LK, Jabs R, and Walz W. GABAA receptor agonists modulate K+ currents in adult hippocampal glial cells in situ. Glia 26: 129–138, 1999.[CrossRef][Web of Science][Medline]
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