- Email: [email protected]
Ethanol effects on electrophysiological properties of astrocytes in striatal brain slices
Neuropharmacology 51 (2006) 1099e1108 www.elsevier.com/locate/neuropharm
Ethanol effects on electrophysiological properties of astrocytes in striatal brain slices Louise Adermark, David M. Lovinger* Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, NIAAA/NIH, 5625 Fishers Lane, TS-13, Bethesda, MD 20892, USA Received 8 March 2006; received in revised form 2 May 2006; accepted 25 May 2006
Abstract Ethanol (EtOH) is known to alter neuronal physiology, but much less is known about the actions of this drug on glial function. To this end, we examined acute effects of ethanol on resting and voltage-activated membrane currents in striatal astrocytes using rat brain slices. Ten minutes exposure to 50 mM EtOH reduced slope conductance by 20%, increased input resistance by 25% and decreased capacitance by 38% but did not affect resting membrane potential. Current generated by a hyperpolarizing pulse was inhibited in a concentration dependent manner in passive astrocytes, while no significant EtOH effect was observed in complex astrocytes or neurons. The EtOH effect was blocked when intracellular KCl was replaced with CsCl, but not during chelation of intracellular calcium with BAPTA. During blockage of gap junction coupling with high intracellular CaCl2 or extracellular carbenoxolone the EtOH effect persisted but was reduced. Interestingly, EtOH effects were largely irreversible when gap junctions were open, but were fully reversible when gap junctions were closed. Ethanol also reduced the spread to other cells of Lucifer Yellow dye from individual glia filled via the patch pipette. These data suggest that EtOH inhibits a calcium-insensitive potassium channel, most likely a passive potassium channel, but also affects gap junction coupling in a way that is sustained after ethanol withdrawal. Astrocytes play a critical role in brain potassium homeostasis, and therefore EtOH effects on astrocytic function could influence neuronal activity. Published by Elsevier Ltd. Keywords: Alcohol; Astroglia; Gap junction; GFAP; Patch clamp; Whole cell
1. Introduction Astrocytes are the most numerous cell type of the glia family and play an active role in neuronal development and function. Glial cells can control synaptogenesis by secreting factors that regulate synapse assembly and functional maturation (Freeman, 2005, 2006), and it has been suggested that synapses cannot develop correctly without astrocytes (Slezak and Pfrieger, 2003). There is a dynamic two-way communication between astrocytes and neurons at the synapse. Astrocytes express numerous receptors, uptake systems and ion channels that are activated by neuronal activity (Bordey and Sontheimer, 2000; D’Ascenzo et al., 2004; Parkerson and Abbreviations: EtOH, ethanol; GFAP, glial fibrillary acidic protein. * Corresponding author. Tel.: þ1 301 443 2445; fax: þ1 301 480 0466. E-mail address: [email protected] (D.M. Lovinger). 0028-3908/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.neuropharm.2006.05.035
Sontheimer, 2004; Walz, 2000), but astrocytes may also modulate synaptic transmission (Haydon, 2000; Nedergaard, 1994; Nedergaard et al., 2003). Astrocytes are interconnected through gap junction channels, and a functional astrocytic syncytium has been suggested to be important for neuronal communication and synaptic plasticity (Cotrina et al., 1998; Giaume and Venance, 1998; Janigro et al., 1997). Electrophysiological recordings of astrocytes in brain slices from hippocampus and cerebral cortex suggest coexistence of a variety of astrocytes with contrasting properties, often categorized as ‘‘passive’’ or ‘‘complex’’ (Anderova et al., 2004; Bekar et al., 2005; Isokawa and McKhann, 2005; Wallraff et al., 2004; Zhou et al., 2006). Ethanol (EtOH), which is one of the most commonly used psychoactive drugs, affects hormone- and neurotransmitteractivated receptors and signal transduction in ways that lead to short-term changes in cellular functions and long-term
1100
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
changes in gene expression (Diamond and Gordon, 1997; Lovinger, 1997). The striatum is a major target for the actions of drugs of abuse. Basal ganglia/limbic striatal and thalamocortical circuits are involved in craving and loss of control in alcohol abuse and dependence as alcohol-associated cues activate the brain reward system and motivate alcohol intake (Braus et al., 2001; Grusser et al., 2004; Modell et al., 1990). Recently, it was suggested that the striatum is a brain center for habit formation. Accordingly, it has been proposed that the dorsal striatum is likely to be involved in advanced stages of addiction when drug use progresses towards a habitual and eventually a compulsive pathology (Gerdeman et al., 2003). Acute alcohol effects on astrocytes include changes in morphology, cell volume, uptake and release of amino acids, gap junction communication and energy metabolism (Adermark et al., 2004; Allansson et al., 2001; Fonseca et al., 2001; Haghighat et al., 1999; Othman et al., 2002). Given that astrocytes are important for brain function (Fellin and Carmignoto, 2004), EtOH-mediated effects on astrocyte physiology could have pronounced effects on neurotransmission and excitability, and neuronal health in the CNS. We hypothesize that EtOH effects on cell volume, morphology and function could be connected to EtOH modulation of astrocyte ion homeostasis. EtOH effects on ion channel function and other electrophysiological parameters might contribute to these actions. The latter hypothesis was tested by investigating acute effects of EtOH on resting and voltage-activated membrane currents using whole-cell patch clamp recordings from striatal astrocytes in rat brain slices. The cells were categorized as passive or complex, and their identity was verified post recording by immunocytochemistry. 2. Methods 2.1. Tissue preparation of brain slices Striatal slices were prepared from 15e21-day-old SpragueeDawley rats. The animals were deeply anesthetized with halothane (Sigma, St. Louis, MO) and decapitated. The brains were quickly removed and placed in icecold modified artificial cerebrospinal fluid (aCSF) containing (in mM); 194 sucrose, 30 NaCl, 4.5 KCl, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4 and 10 D-glucose, saturated with oxygen. After a 5 min equilibration period, the brain tissue was blocked at the anterior and posterior ends and attached with cyanoacrylate to a Teflon pad. The tissue was completely submerged into ice-cold modified aCSF and sectioned coronally in 350 mm thick slices with a VibratomeÒ series 1000 sectioning system (Technical Products International Inc, O’Fallon, MO). Brain slices were allowed to equilibrate for at least 1 h at room temperature in normal aCSF containing (in mM); 124 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4 and 10 D-glucose, and continuously bubbled with a mixture of 95% O2/5% CO2 gas.
2.2. Electrophysiology of brain slices One hemisphere of a striatal brain slice was transferred to a recording chamber connected to a gravity-assisted superfusion system. The slice was held to the bottom of the chamber with silver wires and superfused at a constant rate of 2 ml/min by aCSF bubbled with a mixture of 95% O2/5% CO2 gas. The brain slice was viewed with a water immersion objective (40 NA 0.80, LUMPlan Fl) attached to an upright Olympus BX50WI microscope, and glial cells were identified on a monitor attached to a CCD72 camera
(MTI, Tustin, CA). Patch pipettes made from thin-walled borosilicate glass (TW150F-4; World Precision Instruments, Sarasota, FL) were pulled to micrometer diameter tips (Model P-97; Sutter Instrument Company, Novato, CA) with a typical resistance of 2e6 MU. Currents were measured in conventional ruptured-patch whole-cell mode using pipettes filled with a solution that consisted of (in mM); KCl 130, MgCl2 2, HEPES 10, EGTA 5, Na-ATP 2, CaCl2 0.5, with pH set to 7.3 with KOH (as in Kronenberg et al., 2005). Other internal solutions used contained CsCl as a substitute for KCl, or 10 mM BAPTA in exchange for EGTA and CaCl2. To block gap junction coupling, the intracellular calcium concentration ([Ca2þ]i) was increased to 2 mM with CaCl2 in some recordings (Enkvist and McCarthy, 1994). Osmolarity of internal solutions and aCSF were set to 300 mmol/kg and 314 mmol/kg respectively, with sucrose. An axopatch 200B amplifier (Axon Instruments, Foster City, CA) was used for signal amplication and as a low-pass filter (2 kHz, bessel filter). The preamplifier head stage and pipette holder were mounted onto a Burleigh PCS5000 micromanipulator (EXFO Life Sciences Group, Ontario, Canada). A Digidata 1322A (Axon Instruments) interfaced to a PC compatible computer digitized the signal on line at 10e100 kHz. The PClamp 8.2 software package (Axon Instruments) was used for data acquisition. Input resistance was measured in response to a 5 mV pulse with a 10 ms duration and resting membrane potential was determined by finding the membrane potential at which the steady-state current level was zero (0 current level). Apparent whole-cell capacitance was measured from capacitive transients generated by the 5 mV pulse. The response to weak hyperpolarization was evaluated by subjecting cells to 150 ms pulses of 20 mV from the holding potential of 70 mV delivered 1/10 s. To determine the presence or absence of voltagegated currents, membrane potential was stepped for 250 ms from the holding potential of 70 mV across a range of hyper- and depolarizing potentials from 130 to 60 mV, in 10 mV increments. Current/voltage (I/V) relationships were evaluated at steady state near the end of the 250 ms step. After establishing the whole cell recording, cells were allowed to stabilize for 15e20 min, and a stable current/voltage relationship was a criterion before slices were exposed to EtOH. I/V relationships and responses to 20 mV hyperpolarizing pulses were examined before EtOH application, after 10 min of EtOH exposure, and 10, 20 and 30 min after returning to normal aCSF. Experiments were discarded if the series resistance changed by more than 20% after initiation of data collection, or if resting membrane potential was more depolarized than 40 mV. EtOH was dissolved in aCSF to final concentrations of 10, 25, 50 and 100 mM. Octanol was dissolved in DMSO to 3.1% and diluted in aCSF to a final concentration of 1 mM. Carbenoxolone was dissolved to 150 mM in H2O and diluted in aCSF to a final concentration of 100 mM. All recordings were performed at 30 C. Data are presented as percentage of control before and during EtOH exposure. To evaluate the impact of ethanol on spread of Lucifer Yellow dye from a single astrocyte, slices were incubated in aCSF (control) or 50 mM EtOH for 10 min before whole cell recordings with an internal solution containing 0.05 mg/ml Lucifer Yellow were established. Patch clamped cells were held at 70 mV for 10 min during which time the dye was allowed to spread throughout the syncytium. The number of stained cells was calculated by manually counting in focus cells on 2D images captured with an Axiovert 200 epifluorescence microscope using a 10, NA 0.30 Plan-NEOFLUAR objective and an AxioCam MR monochrome camera (Carl Zeiss Inc., Thornwood, NY).
2.3. Immunohistochemistry For morphological and immunohistochemical analysis of patch clamped cells, 0.01 mg/ml Lucifer Yellow (Molecular Probes, Leiden, Netherlands) was added to the internal solution. Slices were fixed overnight in 4% formaldehyde in 0.1 M phosphate buffered saline (PBS) at 4 C, then washed and kept in 0.1 M PBS at 4 C. To improve antibody penetration and decrease autofluorescence, brain slices were delipidated using an ethanol gradient (70, 80, 95, 100, 95, 80, 70 mM) in 15 min steps, and rinsed in PBS. Slices were incubated in PBS containing 0.2% Triton X-100 (PBS-T) for 1 h, then subjected to 1% Triton X-100 for one additional hour before blocking with 5% BSA in PBS-T overnight. Primary antibodies against glial fibrillary acidic protein (GFAP) (mouse monoclonal (1:200), Chemicon, Temecula, CA), microfilament associated
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108 protein (MAP) (rabbit monoclonal 1:200, Chemicon) or OLIG1 (rabbit monoclonal 1:200, Chemicon), which identify astroglial cells, neurons and oligodendrocytes, respectively, were diluted in PBS-T and stained for 48e72 h. Brain slices were rinsed 3 1 h in PBS-T, and incubated with secondary antibodies [Alexa 568 anti mouse (1:1000), Alexa 360 anti mouse (1:1000), Alexa 555 anti rabbit (1:500)] for at least 15 h. Brain slices were washed 3 1 h in PBS-T, then washed with TBS and mounted with fluorescent mounting medium (Component A, Molecular Probes, Eugene, Or) in secure-sealÔ spacers (Molecular Probes) attached to coverslips (No. 1½; 24 60 mm, Corning, NY) and sealed with coverglass No. 1 (Corning) and nail polish before being viewed with an Axiovert 200 epifluorescence microscope with an AxioCam MR monochrome camera (Carl Zeiss Inc.) [10, NA 0.30 Plan-Neofluar; 32, NA 0.40 Ph1 FL-D; 100, oil immersion, NA 1.3 Plan-Neofluar]. Lucifer Yellow was excited at 480/30, dichroic mirror 505DCLP, and emitter D535/40m. Alexa 568 was excited at D540/25, dichroic was 565 DCLP and emitted light was filtered through a bandpass filter D605/55m. AxioVision 3.1 software was used to collect data, and figures were assembled using Adobe Photoshop.
2.4. Statistics Parametric description of baseline membrane properties, such as those presented in Table 1, are described as mean absolute values with the 95% confidence interval (CI). Due to the variation in baseline membrane properties (i.e. IR, Cm, etc.) it was not possible to compare ethanol effects across cells. Thus, we employed a repeated measures design in which ethanol effects on membrane properties were compared within a given cell to the measurements made before ethanol exposure. Data from these experiments are presented as the mean percent difference relative to control with CI. The level of significance is presented in each figure. Statistical comparisons were with the Student’s t-test.
3. Results 3.1. Electrophysiological properties EtOH effects on astrocytes were evaluated in a total of 102 cells. The astrocytes showed variability in input resistance (range 5e180 MU), as well as in the apparent whole-cell capacitance values (range 1e297 pF). These measures are undoubtedly distorted by the presence of a gap junction-connected astrocyte syncytium. Astrocytes exhibited average resting membrane potentials (0 current levels) of 67 1.5 mV (n ¼ 64), which was close to the holding potential of 70 mV used in our voltage-clamp experiments. The cells were divided into two groups based on electrophysiological properties (Fig. 1). Of the analyzed cells, 87% exhibited a relatively linear I/V relationship and are referred to as passive astrocytes. Passive astrocytes exhibited a large time- and voltage independent conductance. The currents observed in the cells were almost certainly due to the combination of potassium and gap junction channel conductances, as almost no conductance was detected in cells loaded with CsCl in combination Table 1 Intrinsic properties of passive striatal astrocytes before and after 10 min exposure to 50 mM EtOH
IR (MU) Vm (mV) Cm (pF)
Control (n ¼ 15)
EtOH (% of control, n ¼ 15)
42 (CI 7.3) 64 (CI 3.0) 99 (CI 51)
125 (CI 13)*** 102 (CI 3.3)n.s. 72 (CI 17)*
***p < 0.001; *p < 0.05; n.s., Not Specified.
1101
with high [Ca2þ]i (Fig. 1). We did not observe any evidence of a fast inward current upon depolarization in passive astrocytes. Complex astrocytes expressed a combination of timeand voltage-activated current, as well as a large passive current (Fig. 1). The majority of the time- and voltage-dependent current was outward, but some cells exhibited fast inward currents upon depolarization. 3.2. EtOH effects on astrocytic ion currents Ethanol was applied to slices via bath perfusion and we measured responses to brief hyperpolarizing pulses (20 mV, 150 ms) delivered 1/10 s, and currents activated by de- and hyperpolarizing pulses (130 to 60) in 10 mV increments from the holding potential (70 mV). In passive astrocytes, EtOH reduced membrane conductance. This was most readily observed as a decrease in the amplitude of current activated by hyperpolarizing pulses (Fig. 2). Responses to these pulses stabilized following initiation of whole-cell recording, and subsequent application of 25 mM EtOH produced a decrease in current amplitude that developed gradually over the first 15 min of drug application. With continuous application of 25 mM EtOH we observed maximal inhibition of 27 5.5% after 40 min. According to chamber volume and flow rate, the buffer in the chamber would be completely exchanged within w1 min, and distribution of EtOH throughout the slice preparation should be fairly rapid given the amphipathic nature of this compound. Thus, the EtOH effects are delayed by a matter of a few minutes relative to the equilibration of alcohol in the slice. The amplitude of current produced by hyperpolarization did not return to pre-EtOH baseline levels following removal of alcohol from the preparation, even with perfusion times as long as 30 min (Fig. 2b). Cells not treated with EtOH exhibited a constant response to the hyperpolarizing current as long as the series resistance was stable (Fig. 2b). EtOH inhibited current generated by hyperpolarization and decreased slope conductance in passive astrocytes to a similar extent. The decrease in current amplitude was concentration dependent (Fig. 3), and 10 min exposure to 10, 25, 50 or 100 mM EtOH significantly reduced slope conductance in passive astrocytes to 88 6.0% (n ¼ 11), 83 6.7% (n ¼ 11), 79 5.9% (n ¼ 13) and 72 9.8% (n ¼ 10) of control values, respectively. Ion current in the presence of EtOH still lacked any time dependence, and retained the linear I/V relationship (Fig. 1). However, EtOH increased input resistance and decreased apparent cell capacitance (Table 1). Resting membrane current was not significantly affected by EtOH, which might be due to the fact that mean resting membrane potential is close to the holding potential (67 mV vs. 70 mV). After 10 min exposure to 10 mM, 25 mM, 50 mM or 100 mM EtOH resting membrane current in passive astrocytes was 100 4.0% (n ¼ 10), 96 5.5% (n ¼ 11), 96 6.2% (n ¼ 14) and 101 8.3% (n ¼ 9) of control. In complex astrocytes and neurons resting membrane current was 97 3.2% (n ¼ 12), and 94 6.6% (n ¼ 11) of control respectively, after 10 min exposure to 50 mM EtOH.
1102
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Fig. 1. Ion currents in passive and complex striatal astrocytes. Representative traces showing membrane current patterns and current/voltage relationship of one passive astrocyte (a), one complex astrocyte (b), and one passive cell in which intracellular KCl was replaced with CsCl þ 2 mM CaCl2 (c), before and after 10 min exposure to 50 mM EtOH. EtOH did not alter the lack of time-dependence or the linear shape of the I/V relationship, but reduced conductance, in passive astrocytes. Passive current was greatly reduced in cells filled with CsCl, and CaCl2 indicating that the major conductances in passive astrocytes are generated by potassium channels and gap junctions. EtOH had no effect on the remaining current in the CsCl þ CaCl2-filled cells. Membrane current patterns were measured in response to hyper- and depolarizing pulses given in 10 mV increments from a holding potential of 70 mV. I/V relationships plotted at the right were constructed from current measurements made at steady-state just before the end of the 250 ms voltage pulse.
In complex astrocytes, 10 min exposure to 50 mM EtOH did not affect current generated by hyperpolarization (96 6.3%, n ¼ 8) (Fig. 3). Likewise, 50 mM EtOH had no effect on currents generated by hyperpolarization in striatal neurons (102 7.3%, n ¼ 12) (Fig. 3). 3.3. Contribution of Kþ and gap junction conductances to the EtOH effect We examined the effects on the EtOH-induced inhibition of a number of manipulations designed to reduce particular cellular conductances. Blockage of potassium channels by substituting intracellular CsCl for KCl increased input resistance to 66 12 MU (n ¼ 10), decreased apparent capacitance (10 3.7 pF, n ¼ 10) but had no significant effect on membrane potential 57 5.6 mV (n ¼ 10). Substitution of KCl with CsCl in the patch pipette completely blocked the EtOH-mediated decrease in current amplitude (Fig. 4), and slope conductance (95 5.1%, n ¼ 11, 50 mM EtOH). Intracellular loading with 10 mM of the calcium chelator BAPTA
did not affect intrinsic properties in passive astrocytes. EtOH (50 mM) reduced hyperpolarization-induced current (Fig. 4), and decreased slope conductance by 23 5.4% under this condition (n ¼ 9), indicating that EtOH effects on astrocytes occur even in the presence of very low [Ca2þ]i. We attempted to block gap junction coupling via extracellular application of 1 mM octanol or 100 mM carbenoxolone, or intracellular loading of 2 mM CaCl2. Gap junctions were considered to be closed if input resistance markedly increased and apparent capacitance decreased within a 20 min time frame. None of the blockers used had a 100% success rate. Increased intracellular calcium produced changes indicative of gap junction closer in 64% of the cells examined. In cells where the blockers were effective input resistance increased (109 19 MU for octanol; 90 27 MU for carbenoxolone; 98 19 MU for intracellular CaCl2) and apparent capacitance decreased (25 12 pF; 22 12 pF; 15 6.6 pF for octanol, carbenoxolone, and intracellular CaCl2, respectively). Octanol and carbenoxolone shifted resting membrane potential to a more positive potential (43 7.9 mV and 46 12 mV,
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
1103
Fig. 2. Ethanol inhibition of current produced by hyperpolarization in passive astrocytes. (a) Currents generated by hyperpolarizing pulses of 20 mV (from a 70 mV holding potential) were allowed to stabilize following initiation of whole-cell recordings, and subsequent application of 25 mM EtOH produced a decrease in current amplitude that developed gradually over the first 15 min of drug application. Data are averaged values from seven passive astrocytes, and are presented as mean percent of control with 95% CI. (b) EtOH inhibition of current in response to hyperpolarization was largely irreversible in KCl filled astrocytes. The decrease in current amplitude mediated by 10 min exposure to 50 mM EtOH still remained after 30 min washout with EtOH-free solution. Current generated by hyperpolarization was stable in astrocytes not exposed to EtOH. Each measurement point is based on data from 15 cells, with 95% CI.
respectively), while 2 mM CaCl2 had no effect (61 8.3 mV). During blockage of gap junction coupling in cells treated with 1 mM octanol the inhibition by EtOH was intact (Fig. 4), and 50 mM EtOH reduced slope conductance to 83 11% of control values (n ¼ 10). Treatment with carbenoxolone or 2 mM CaCl2 did not eliminate the effects of EtOH, but the alcohol-induced decrease in the response to the hyperpolarizing pulse was significantly reduced (Fig. 4). Slope conductance was 88 5% (n ¼ 8) and 86 4% (n ¼ 13), respectively, of control values in the presence of 50 mM ethanol in these cells. We noted that when cells were treated with carbenoxolone or increased intracellular
CaCl2, the EtOH effect was fully reversible within 10 min upon washout of the alcohol (Fig. 5), whereas EtOH effects were largely irreversible under all of the other experimental conditions (with the exception of CsCl-filled cells where EtOH had no effect) (compare to Fig. 2). In those cells in which elevated intracellular Ca2þ had little effect on input resistance and capacitance EtOH effects were sustained during washout, indicating that the effect is connected to a reduction in gap junction coupling and not caused by the high [Ca2þ]i. EtOH did not affect conductance in cells loaded with CsCl and 2 mM CaCl2 (Fig. 1). Slope conductance was 100 3.5% of control, and current generated by hyperpolarization was 100 2.3% of control value (n ¼ 4). Spreading of Lucifer Yellow decreased by 46% in slices preincubated with 50 mM EtOH for 10 min before establishing whole cell recordings (Fig. 6). The number of stained cells was 507 287 (n ¼ 12) for control and 276 191 (n ¼ 8) in the EtOH treated slices ( p < 0.05). 3.4. Immunohistochemistry
Fig. 3. Ethanol inhibition is concentration dependent, and specific to passive astrocytes. Ten minutes exposure to 10, 25, 50 or 100 mM inhibited current generated by hyperpolarization in passive astrocytes in a concentration dependent manner, but did not affect hyperpolarization-induced current in complex astrocytes or neurons. Data are presented as percent of control with 95% CI. **p < 0.01, *p < 0.05.
Despite the use of high concentrations of Triton X-100 and long incubation times, antibody penetration into slices was poor. As patch clamped cells in most recordings were positioned deep within the slice, only a fraction (w20%) of the slices examined electrophysiologically were satisfactory stained. However, in the cases in which slices containing Lucifer Yellow stained cells were successfully labeled immunohistochemically the Lucifer-filled cells were positive for GFAP (n ¼ 25) (Fig. 7). Co-localization of Lucifer Yellow and OLIG1-, or MAP staining was not found indicating that none of the patch clamped cells were oligodendrocytes or neurons (based on 35 slices). No morphological differences between passive and complex astrocytes could be verified in the set of data presented here, mainly due to the fact that only a small number of complex cells were encountered, and many of these were not successfully labeled.
1104
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Fig. 4. (a) Graphs summarizing EtOH effects on the hyperpolarization-induced current during the indicated experimental manipulations. The EtOH effect was lost when CsCl was substituted for KCl in the internal solution, but remained in cells filled with 10 mM BAPTA, suggesting that ethanol affects calcium insensitive potassium channels. The EtOH effect was sustained during blockage of gap junctions with 1 mM octanol, but was significantly reduced when channels were closed with 100 mM carbenoxolone or intracellular loading with 2 mM CaCl2. Data are presented as mean percent of control values with 95% CI. #p < 0.05, ##p < 0.01, **p < 0.01, ***p < 0.001. (b) Representative traces showing currents generated by hyperpolarization in passive astrocytes before and during exposure to 50 mM EtOH. Astrocytes were held at 70 mV and subject to a hyperpolarizing pulse of 20 mV for 250 ms. Exposure to 1 mM octanol decreased the amplitude of the response in all cells, but EtOH reduced it further.
4. Discussion EtOH decreased capacitance, inhibited current in response to hyperpolarization and decreased slope conductance in passive astrocytes examined in rat striatal slices. The EtOH effect was blocked when KCl was replaced with CsCl in the internal solution, but not during chelation of intracellular calcium with BAPTA, suggesting that ethanol affects a calcium-insensitive potassium channel, most likely a passive potassium channel. EtOH did not affect currents generated by a hyperpolarizing pulse in complex astrocytes or neurons. The spread of Lucifer Yellow from single dye-filled cells revealed an extensively coupled astrocyte syncytium in the
striatum. Although the number of stained cells varied markedly from slice-to-slice, dye spreading was significantly reduced in brain slices pre-exposed to EtOH, indicating that EtOH acutely decreases gap junction dye permeability. Dye spreading in the striatum has previously been described between rat astrocytes (Hamon et al., 2002), while no dye spreading was reported between mouse striatal astrocytes (Rufer et al., 1996). However, the mouse astrocytes examined by Rufer et al. (1996) expressed connexin-43 and currents from surrounding cells could be detected, suggesting that there is a functional network. It is possible that the low temperature used in the Rufer et al. (1996) study might have contributed to the lack of dye spreading (Bukauskas and Weingart, 1993).
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Fig. 5. The decrease in current amplitude mediated by 10 min exposure to 50 mM EtOH was irreversible in KCl filled astrocytes but returned to baseline level within 10 min after beginning the washout of EtOH under conditions in which gap junction coupling was blocked by carbenoxolone or intracellular loading with 2 mM CaCl2. Graph describes current amplitude after 10 min exposure to 50 mM EtOH, and after 10 min rinsing with aCSF. Data are presented as mean percent of control values with 95% CI.
The increase in input resistance and decrease in capacitance detected during EtOH exposure might also be directly connected to a change in gap junction permeability. Apparent capacitance gives an indication of the membrane surface area of the cell and the decrease in this value could be a sign that the connection to other astrocytes has been reduced. Theoretically (Ohms law), an increase in input resistance would be expected when gap junctional currents are reduced. This has also been shown in rat slices from the hippocampus and striatum (Blomstrand et al., 2004; Hamon et al., 2002), while contradictory results were described for mouse astrocytes (Wallraff et al., 2004). However, in the latter study gap junctional blockers were only added for 3 min, and based on the thickness of the slices and the slow penetration of drugs into such slices it is possible that this duration of exposure
1105
is not sufficiently long to shut down enough gap junction channels to increase input resistance (Wallraff et al., 2004). All of the gap junction blockers that we employed in the present study produced an increase in input resistance, and thus this measure appears to be a reliable indicator of a decrease in gap junctional coupling. A decrease in dye spreading during EtOH exposure has previously been reported between rat astrocytes in primary astrocyte cultures (Adermark et al., 2004). The significant reduction in EtOH effect during blockage of gap junctions with carbenoxolone or increased [CaCl2]i gives a further indication that gap junction coupling is inhibited by EtOH. When we attempted to block gap junctions with octanol, the EtOH effect was not significantly reduced. The lack of effect of octanol could be explained by incomplete blockage of gap junction coupling, or could be due to nonspecific effects of octanol (Marcet et al., 2004; Swenson and Narahashi, 1980). Octanol treated cells showed a shift to a more positive resting membrane potential, and the variability in other membrane properties was larger in this treatment group in comparison to other treatments used. Thus, octanol appears to produce disturbances in cell physiology beyond those expected from simple closure of gap junctions. Carbenoxolone also produced a positive shift in resting membrane potential, but the series resistance in these experiments was more stable. Blockage of gap junctions with high intracellular calcium has been reported to be reversed during treatment of cells with high extracellular potassium (55 mM) or 0.4 mM kainate lasting for 10s of minutes (Enkvist and McCarthy, 1994). This effect was attributed to depolarization. However, depolarization was quite prolonged in this study, and both the high potassium and kainate treatments change the ionic balance across the cell membrane, as prolonged kainate would likely increase extracellular Kþ. Thus, it is not clear that the reversal of Ca2þ inhibition is solely due to depolarization. We did not observe any reversal of the effects of high intracellular Ca2þ in our experiments during brief depolarization to potentials of up to þ60 mV. The glial membrane properties were also quite stable in the presence of the high internal calcium. Thus, we believe that raising intracellular calcium is a reliable method for specifically eliminating gap junction coupling.
Fig. 6. The number of cells passively stained with Lucifer Yellow (0.05 mg/ml) was reduced by 46% in slices exposed to 50 mM EtOH for 10 min before establishing whole cell recordings. (a) Control slice; (b) stained cells in a slice exposed to 50 mM EtOH. Scale bar is 200 mm.
1106
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Fig. 7. Wide field images of cells immunoreactive for GFAP (a, c), co-labeled with Lucifer Yellow (b, d), showing that patch clamped cells were astrocytes. The concentration of Lucifer Yellow was low to prevent spreading to surrounding cells. We did not detect any Lucifer Yellow filled cells that stained positive for the oligodendrocyte marker OLIG1 or the neuronal marker MAP (based on evaluation of 35 brain slices). Scale bar is 20 mm.
EtOH effects on intrinsic membrane properties, current generated by hyperpolarization, and slope conductance were largely irreversible when gap junctions were open, but returned to baseline levels after washout of EtOH under two conditions that appeared to decrease gap junctional coupling, namely carbenoxolone or high intracellular calcium treatments. These findings suggest that EtOH affects gap junction coupling in a way that is sustained during EtOH withdrawal. Interestingly, Wentlandt and co-workers showed that the EtOH induced decrease in gap junction coupling between P19 cells did not recover after a 24-h withdrawal period (Wentlandt et al., 2004). The authors suggest that EtOH metabolism or secondmessenger activation after chronic EtOH exposure leads to functional closure of gap junctions. Our data indicate that the irreversible EtOH effect is induced within minutes. The complete blockade of EtOH effects in CsCl-filled cells is surprising. This manipulation should block potassium conductances, but would not necessarily alter the function of gap junctions. We did observe a decrease in apparent capacitance in the presence of intracellular CsCl indicating that this treatment alone might be sufficient to close gap junctions, and thus occlude both effects of EtOH. However, the conductance in Csþ loaded cells was still high, suggesting that gap junctions are not fully closed. Gap junctions formed by the connexin 43 protein (the major form present in astrocytes; Dermietzel et al., 1991) exhibit at least three different states; closed, open and residual (Valiunas et al., 1997), and single channel conductance
has been shown to depend on whether KCl or Cs-aspartate is present in the internal solution (Valiunas et al., 1997). Furthermore, it has been proposed that hemichannels formed by connexin 50 are regulated by monovalent cations (Srinivas et al., 2006), and this could be the mechanism through which internal Csþ also affects gap junction channels formed by connexin 43. In this context, it is interesting that the EtOH-mediated decrease in gap junction coupling in astrocyte cultures was blocked during manipulations that would affect potassium and sodium homeostasis (Adermark et al., 2004). It is possible that Csþ or the decrease in intracellular potassium affects the channel properties or gating in a way that interacts with the effect of EtOH, but it is also possible that the alcohol effect on gap junctions is somehow secondary to the change in potassium conductance. EtOH effects on passive potassium conductance and/or gap junction conductance could be mediated by a number of mechanisms. Ethanol-induced disturbances in intracellular signaling by PKC, and EtOH effects on the cytoskeleton, have been observed, and both of these processes are linked to the function of gap junction-forming connexins (Li et al., 2005; Newton and Messing, 2006; Shumilla et al., 2005; Zvalova et al., 2004). Future studies may elucidate the mechanisms underlying the EtOH effects. The data presented here suggest that EtOH effects on striatal astrocytes involve calcium insensitive potassium channels, and that EtOH modulates gap junction channels in a way that
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
is sustained during EtOH withdrawal. Astrocytes are important for maintaining potassium homeostasis, and effects of EtOH reported in the present study could produce secondary effects on neurotransmission and plasticity. Acknowledgments This work was sponsored by the Division of Intramural Clinical and Basic Research, NIAAA, NIH. We thank Dr. Lixian Dong for helpful discussions of gap junction pharmacology. References Adermark, L., Olsson, T., Hansson, E., 2004. Ethanol acutely decreases astroglial gap junction permeability in primary cultures from defined brain regions. Neurochem. Int. 45, 971e978. Allansson, L., Khatibi, S., Olsson, T., Hansson, E., 2001. Acute ethanol exposure induces [Ca2þ]i transients, cell swelling and transformation of actin cytoskeleton in astroglial primary cultures. J. Neurochem. 76, 472e479. Anderova, M., Antonova, T., Petrik, D., Neprasova, H., Chvatal, A., Sykova, E., 2004. Voltage-dependent potassium currents in hypertrophied rat astrocytes after a cortical stab wound. Glia 48, 311e326. Bekar, L.K., Loewen, M.E., Cao, K., Sun, X., Leis, J., Wang, R., Forsyth, G.W., Walz, W., 2005. Complex expression and localization of inactivating Kv channels in cultured hippocampal astrocytes. J. Neurophysiol. 93, 1699e1709. Blomstrand, F., Venance, L., Siren, A.L., Ezan, P., Hanse, E., Glowinski, J., Ehrenreich, H., Giaume, C., 2004. Endothelins regulate astrocyte gap junctions in rat hippocampal slices. Eur. J. Neurosci. 19, 1005e1015. Bordey, A., Sontheimer, H., 2000. Ion channel expression by astrocytes in situ: comparison of different CNS regions. Glia 30, 27e38. Braus, D.F., Wrase, J., Grusser, S., Hermann, D., Ruf, M., Flor, H., Mann, K., Heinz, A., 2001. Alcohol-associated stimuli activate the ventral striatum in abstinent alcoholics. J. Neural Transm. 108, 887e894. Bukauskas, F.F., Weingart, R., 1993. Temperature dependence of gap junction properties in neonatal rat heart cells. Pflugers Arch. 423, 133e139. Cotrina, M.L., Lin, J.H., Alves-Rodrigues, A., Liu, S., Li, J., AzmiGhadimi, H., Kang, J., Naus, C.C., Nedergaard, M., 1998. Connexins regulate calcium signaling by controlling ATP release. Proc. Natl. Acad. Sci. U.S.A. 95, 15735e15740. D’Ascenzo, M., Vairano, M., Andreassi, C., Navarra, P., Azzena, G.B., Grassi, C., 2004. Electrophysiological and molecular evidence of L(Cav1), N- (Cav2.2), and R- (Cav2.3) type Ca2þ channels in rat cortical astrocytes. Glia 45, 354e363. Dermietzel, R., Hertberg, E.L., Kessler, J.A., Spray, D.C., 1991. Gap junctions between cultured astrocytes: immunocytochemical, molecular, and electrophysiological analysis. J. Neurosci. 11, 1421e1432. Diamond, I., Gordon, A.S., 1997. Cellular and molecular neuroscience of alcoholism. Physiol. Rev. 77, 1e20. Enkvist, M.O., McCarthy, K.D., 1994. Astroglial gap junction communication is increased by treatment with either glutamate or high Kþ concentration. J. Neurochem. 62, 489e495. Fellin, T., Carmignoto, G., 2004. Neurone-to-astrocyte signalling in the brain represents a distinct multifunctional unit. J. Physiol. 559, 3e15. Fonseca, L.L., Alves, P.M., Carrondo, M.J., Santos, H., 2001. Effect of ethanol on the metabolism of primary astrocytes studied by (13)C- and (31)PNMR spectroscopy. J. Neurosci. Res. 66, 803e811. Freeman, M.R., 2005. Glial control of synaptogenesis. Cell 120, 292e293. Freeman, M.R., 2006. Sculpting the nervous system: glial control of neuronal development. Curr. Opin. Neurobiol. 16, 119e125. Gerdeman, G.L., Partridge, J.G., Lupica, C.R., Lovinger, D.M., 2003. It could be habit forming: drugs of abuse and striatal synaptic plasticity. Trends Neurosci. 26, 184e192.
1107
Giaume, C., Venance, L., 1998. Intercellular calcium signaling and gap junctional communication in astrocytes. Glia 24, 50e64. Grusser, S.M., Wrase, J., Klein, S., Hermann, D., Smolka, M.N., Ruf, M., Weber-Fahr, W., Flor, H., Mann, K., Braus, D.F., Heinz, A., 2004. Cueinduced activation of the striatum and medial prefrontal cortex is associated with subsequent relapse in abstinent alcoholics. Psychopharmacology (Berl) 175, 296e302. Haghighat, N., McCandless, D.W., Geraminejad, P., 1999. Effect of alcohol on energy storage of primary astrocytes and C6-glioma cells in vitro. Metab. Brain Dis. 14, 149e156. Hamon, B., Glowinski, J., Giaume, C., 2002. Intercellular diffusional coupling between glial cells in slices from the striatum. In: de Vellis, J. (Ed.), Neuroglia in the Aging Brain. Humana Press, Totowa, pp. 99e112. Haydon, P.G., 2000. Neuroglial networks: neurons and glia talk to each other. Curr. Biol. 10, R712eR714. Isokawa, M., McKhann, G.M., 2005. Electrophysiological and morphological characterization of dentate astrocytes in the hippocampus. J. Neurobiol. 65, 125e134. Janigro, D., Gasparini, S., D’Ambrosio, R., McKhann, G., DiFrancesco, D., 1997. Reduction of Kþ uptake in glia prevents long-term depression maintenance and causes epileptiform activity. J. Neurosci. 17, 2813e2824. Kronenberg, G., Wang, L.P., Synowitz, M., Gertz, K., Katchanov, J., Glass, R., Harms, C., Kempermann, G., Kettenmann, H., Endres, M., 2005. Nestin-expressing cells divide and adopt a complex electrophysiologic phenotype after transient brain ischemia. J. Cereb. Blood Flow Metab. 25, 1613e1624. Li, W., Hertzberg, E.L., Spray, D.C., 2005. Regulation of connexin43-protein binding in astrocytes in response to chemical ischemia/hypoxia. J. Biol. Chem. 280, 7941e7948. Lovinger, D.M., 1997. Alcohols and neurotransmitter gated ion channels: past, present and future. Naunyn Schmiedebergs Arch. Pharmacol. 356, 267e 282. Marcet, B., Becq, F., Norez, C., Delmas, P., Verrier, B., 2004. General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels. Br. J. Pharmacol. 141, 905e914. Modell, J.G., Mountz, J.M., Beresford, T.P., 1990. Basal ganglia/limbic striatal and thalamocortical involvement in craving and loss of control in alcoholism. J. Neuropsychiatry Clin. Neurosci. 2, 123e144. Nedergaard, M., 1994. Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science 263, 1768e1771. Nedergaard, M., Ransom, B., Goldman, S.A., 2003. New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci. 26, 523e530. Newton, P.M., Messing, R.O., 2006. Intracellular signaling pathways that regulate behavioral responses to ethanol. Pharmacol. Ther. 109, 227e237. Othman, T., Sinclair, C.J., Haughey, N., Geiger, J.D., Parkinson, F.E., 2002. Ethanol alters glutamate but not adenosine uptake in rat astrocytes: evidence for protein kinase C involvement. Neurochem. Res. 27, 289e296. Parkerson, K.A., Sontheimer, H., 2004. Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes. Glia 46, 419e436. Rufer, M., Wirth, S.B., Hofer, A., Dermietzel, R., Pastor, A., Kettenmann, H., Unsicker, K., 1996. Regulation of connexin-43, GFAP, and FGF-2 is not accompanied by changes in astroglial coupling in MPTP-lesioned, FGF2-treated parkinsonian mice. J. Neurosci. Res. 46, 606e617. Shumilla, J.A., Liron, T., Mochly-Rosen, D., Kendig, J.J., Sweitzer, S.M., 2005. Ethanol withdrawal-associated allodynia and hyperalgesia: agedependent regulation by protein kinase C epsilon and gamma isoenzymes. J. Pain 6, 535e549. Slezak, M., Pfrieger, F.W., 2003. New roles for astrocytes: regulation of CNS synaptogenesis. Trends Neurosci. 26, 531e535. Srinivas, M., Calderon, D.P., Kronengold, J., Verselis, V.K., 2006. Regulation of connexin hemichannels by monovalent cations. J. Gen. Physiol. 127, 67e75. Swenson, R.P., Narahashi, T., 1980. Block of sodium conductance by n-octanol in crayfish giant axons. Biochim. Biophys. Acta. 603, 228e236.
1108
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Valiunas, V., Bukauskas, F.F., Weingart, R., 1997. Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells. Circ. Res. 80, 708e719. Wallraff, A., Odermatt, B., Willecke, K., Steinhauser, C., 2004. Distinct types of astroglial cells in the hippocampus differ in gap junction coupling. Glia 48, 36e43. Walz, W., 2000. Controversy surrounding the existence of discrete functional classes of astrocytes in adult gray matter. Glia 31, 95e103.
Wentlandt, K., Kushnir, M., Naus, C.C., Carlen, P.L., 2004. Ethanol inhibits gap-junctional coupling between P19 cells. Alcohol Clin. Exp. Res. 28, 1284e1290. Zhou, M., Schools, G.P., Kimelberg, H.K., 2006. Development of GLAST(þ) astrocytes and NG2(þ) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive. J. Neurophysiol. 95, 134e143. Zvalova, D., Cordier, J., Mesnil, M., Junier, M.P., Chneiweiss, H., 2004. p38/ SAPK2 controls gap junction closure in astrocytes. Glia 46, 323e333.
Ethanol effects on electrophysiological properties of astrocytes in striatal brain slices Louise Adermark, David M. Lovinger* Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, NIAAA/NIH, 5625 Fishers Lane, TS-13, Bethesda, MD 20892, USA Received 8 March 2006; received in revised form 2 May 2006; accepted 25 May 2006
Abstract Ethanol (EtOH) is known to alter neuronal physiology, but much less is known about the actions of this drug on glial function. To this end, we examined acute effects of ethanol on resting and voltage-activated membrane currents in striatal astrocytes using rat brain slices. Ten minutes exposure to 50 mM EtOH reduced slope conductance by 20%, increased input resistance by 25% and decreased capacitance by 38% but did not affect resting membrane potential. Current generated by a hyperpolarizing pulse was inhibited in a concentration dependent manner in passive astrocytes, while no significant EtOH effect was observed in complex astrocytes or neurons. The EtOH effect was blocked when intracellular KCl was replaced with CsCl, but not during chelation of intracellular calcium with BAPTA. During blockage of gap junction coupling with high intracellular CaCl2 or extracellular carbenoxolone the EtOH effect persisted but was reduced. Interestingly, EtOH effects were largely irreversible when gap junctions were open, but were fully reversible when gap junctions were closed. Ethanol also reduced the spread to other cells of Lucifer Yellow dye from individual glia filled via the patch pipette. These data suggest that EtOH inhibits a calcium-insensitive potassium channel, most likely a passive potassium channel, but also affects gap junction coupling in a way that is sustained after ethanol withdrawal. Astrocytes play a critical role in brain potassium homeostasis, and therefore EtOH effects on astrocytic function could influence neuronal activity. Published by Elsevier Ltd. Keywords: Alcohol; Astroglia; Gap junction; GFAP; Patch clamp; Whole cell
1. Introduction Astrocytes are the most numerous cell type of the glia family and play an active role in neuronal development and function. Glial cells can control synaptogenesis by secreting factors that regulate synapse assembly and functional maturation (Freeman, 2005, 2006), and it has been suggested that synapses cannot develop correctly without astrocytes (Slezak and Pfrieger, 2003). There is a dynamic two-way communication between astrocytes and neurons at the synapse. Astrocytes express numerous receptors, uptake systems and ion channels that are activated by neuronal activity (Bordey and Sontheimer, 2000; D’Ascenzo et al., 2004; Parkerson and Abbreviations: EtOH, ethanol; GFAP, glial fibrillary acidic protein. * Corresponding author. Tel.: þ1 301 443 2445; fax: þ1 301 480 0466. E-mail address: [email protected] (D.M. Lovinger). 0028-3908/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.neuropharm.2006.05.035
Sontheimer, 2004; Walz, 2000), but astrocytes may also modulate synaptic transmission (Haydon, 2000; Nedergaard, 1994; Nedergaard et al., 2003). Astrocytes are interconnected through gap junction channels, and a functional astrocytic syncytium has been suggested to be important for neuronal communication and synaptic plasticity (Cotrina et al., 1998; Giaume and Venance, 1998; Janigro et al., 1997). Electrophysiological recordings of astrocytes in brain slices from hippocampus and cerebral cortex suggest coexistence of a variety of astrocytes with contrasting properties, often categorized as ‘‘passive’’ or ‘‘complex’’ (Anderova et al., 2004; Bekar et al., 2005; Isokawa and McKhann, 2005; Wallraff et al., 2004; Zhou et al., 2006). Ethanol (EtOH), which is one of the most commonly used psychoactive drugs, affects hormone- and neurotransmitteractivated receptors and signal transduction in ways that lead to short-term changes in cellular functions and long-term
1100
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
changes in gene expression (Diamond and Gordon, 1997; Lovinger, 1997). The striatum is a major target for the actions of drugs of abuse. Basal ganglia/limbic striatal and thalamocortical circuits are involved in craving and loss of control in alcohol abuse and dependence as alcohol-associated cues activate the brain reward system and motivate alcohol intake (Braus et al., 2001; Grusser et al., 2004; Modell et al., 1990). Recently, it was suggested that the striatum is a brain center for habit formation. Accordingly, it has been proposed that the dorsal striatum is likely to be involved in advanced stages of addiction when drug use progresses towards a habitual and eventually a compulsive pathology (Gerdeman et al., 2003). Acute alcohol effects on astrocytes include changes in morphology, cell volume, uptake and release of amino acids, gap junction communication and energy metabolism (Adermark et al., 2004; Allansson et al., 2001; Fonseca et al., 2001; Haghighat et al., 1999; Othman et al., 2002). Given that astrocytes are important for brain function (Fellin and Carmignoto, 2004), EtOH-mediated effects on astrocyte physiology could have pronounced effects on neurotransmission and excitability, and neuronal health in the CNS. We hypothesize that EtOH effects on cell volume, morphology and function could be connected to EtOH modulation of astrocyte ion homeostasis. EtOH effects on ion channel function and other electrophysiological parameters might contribute to these actions. The latter hypothesis was tested by investigating acute effects of EtOH on resting and voltage-activated membrane currents using whole-cell patch clamp recordings from striatal astrocytes in rat brain slices. The cells were categorized as passive or complex, and their identity was verified post recording by immunocytochemistry. 2. Methods 2.1. Tissue preparation of brain slices Striatal slices were prepared from 15e21-day-old SpragueeDawley rats. The animals were deeply anesthetized with halothane (Sigma, St. Louis, MO) and decapitated. The brains were quickly removed and placed in icecold modified artificial cerebrospinal fluid (aCSF) containing (in mM); 194 sucrose, 30 NaCl, 4.5 KCl, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4 and 10 D-glucose, saturated with oxygen. After a 5 min equilibration period, the brain tissue was blocked at the anterior and posterior ends and attached with cyanoacrylate to a Teflon pad. The tissue was completely submerged into ice-cold modified aCSF and sectioned coronally in 350 mm thick slices with a VibratomeÒ series 1000 sectioning system (Technical Products International Inc, O’Fallon, MO). Brain slices were allowed to equilibrate for at least 1 h at room temperature in normal aCSF containing (in mM); 124 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4 and 10 D-glucose, and continuously bubbled with a mixture of 95% O2/5% CO2 gas.
2.2. Electrophysiology of brain slices One hemisphere of a striatal brain slice was transferred to a recording chamber connected to a gravity-assisted superfusion system. The slice was held to the bottom of the chamber with silver wires and superfused at a constant rate of 2 ml/min by aCSF bubbled with a mixture of 95% O2/5% CO2 gas. The brain slice was viewed with a water immersion objective (40 NA 0.80, LUMPlan Fl) attached to an upright Olympus BX50WI microscope, and glial cells were identified on a monitor attached to a CCD72 camera
(MTI, Tustin, CA). Patch pipettes made from thin-walled borosilicate glass (TW150F-4; World Precision Instruments, Sarasota, FL) were pulled to micrometer diameter tips (Model P-97; Sutter Instrument Company, Novato, CA) with a typical resistance of 2e6 MU. Currents were measured in conventional ruptured-patch whole-cell mode using pipettes filled with a solution that consisted of (in mM); KCl 130, MgCl2 2, HEPES 10, EGTA 5, Na-ATP 2, CaCl2 0.5, with pH set to 7.3 with KOH (as in Kronenberg et al., 2005). Other internal solutions used contained CsCl as a substitute for KCl, or 10 mM BAPTA in exchange for EGTA and CaCl2. To block gap junction coupling, the intracellular calcium concentration ([Ca2þ]i) was increased to 2 mM with CaCl2 in some recordings (Enkvist and McCarthy, 1994). Osmolarity of internal solutions and aCSF were set to 300 mmol/kg and 314 mmol/kg respectively, with sucrose. An axopatch 200B amplifier (Axon Instruments, Foster City, CA) was used for signal amplication and as a low-pass filter (2 kHz, bessel filter). The preamplifier head stage and pipette holder were mounted onto a Burleigh PCS5000 micromanipulator (EXFO Life Sciences Group, Ontario, Canada). A Digidata 1322A (Axon Instruments) interfaced to a PC compatible computer digitized the signal on line at 10e100 kHz. The PClamp 8.2 software package (Axon Instruments) was used for data acquisition. Input resistance was measured in response to a 5 mV pulse with a 10 ms duration and resting membrane potential was determined by finding the membrane potential at which the steady-state current level was zero (0 current level). Apparent whole-cell capacitance was measured from capacitive transients generated by the 5 mV pulse. The response to weak hyperpolarization was evaluated by subjecting cells to 150 ms pulses of 20 mV from the holding potential of 70 mV delivered 1/10 s. To determine the presence or absence of voltagegated currents, membrane potential was stepped for 250 ms from the holding potential of 70 mV across a range of hyper- and depolarizing potentials from 130 to 60 mV, in 10 mV increments. Current/voltage (I/V) relationships were evaluated at steady state near the end of the 250 ms step. After establishing the whole cell recording, cells were allowed to stabilize for 15e20 min, and a stable current/voltage relationship was a criterion before slices were exposed to EtOH. I/V relationships and responses to 20 mV hyperpolarizing pulses were examined before EtOH application, after 10 min of EtOH exposure, and 10, 20 and 30 min after returning to normal aCSF. Experiments were discarded if the series resistance changed by more than 20% after initiation of data collection, or if resting membrane potential was more depolarized than 40 mV. EtOH was dissolved in aCSF to final concentrations of 10, 25, 50 and 100 mM. Octanol was dissolved in DMSO to 3.1% and diluted in aCSF to a final concentration of 1 mM. Carbenoxolone was dissolved to 150 mM in H2O and diluted in aCSF to a final concentration of 100 mM. All recordings were performed at 30 C. Data are presented as percentage of control before and during EtOH exposure. To evaluate the impact of ethanol on spread of Lucifer Yellow dye from a single astrocyte, slices were incubated in aCSF (control) or 50 mM EtOH for 10 min before whole cell recordings with an internal solution containing 0.05 mg/ml Lucifer Yellow were established. Patch clamped cells were held at 70 mV for 10 min during which time the dye was allowed to spread throughout the syncytium. The number of stained cells was calculated by manually counting in focus cells on 2D images captured with an Axiovert 200 epifluorescence microscope using a 10, NA 0.30 Plan-NEOFLUAR objective and an AxioCam MR monochrome camera (Carl Zeiss Inc., Thornwood, NY).
2.3. Immunohistochemistry For morphological and immunohistochemical analysis of patch clamped cells, 0.01 mg/ml Lucifer Yellow (Molecular Probes, Leiden, Netherlands) was added to the internal solution. Slices were fixed overnight in 4% formaldehyde in 0.1 M phosphate buffered saline (PBS) at 4 C, then washed and kept in 0.1 M PBS at 4 C. To improve antibody penetration and decrease autofluorescence, brain slices were delipidated using an ethanol gradient (70, 80, 95, 100, 95, 80, 70 mM) in 15 min steps, and rinsed in PBS. Slices were incubated in PBS containing 0.2% Triton X-100 (PBS-T) for 1 h, then subjected to 1% Triton X-100 for one additional hour before blocking with 5% BSA in PBS-T overnight. Primary antibodies against glial fibrillary acidic protein (GFAP) (mouse monoclonal (1:200), Chemicon, Temecula, CA), microfilament associated
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108 protein (MAP) (rabbit monoclonal 1:200, Chemicon) or OLIG1 (rabbit monoclonal 1:200, Chemicon), which identify astroglial cells, neurons and oligodendrocytes, respectively, were diluted in PBS-T and stained for 48e72 h. Brain slices were rinsed 3 1 h in PBS-T, and incubated with secondary antibodies [Alexa 568 anti mouse (1:1000), Alexa 360 anti mouse (1:1000), Alexa 555 anti rabbit (1:500)] for at least 15 h. Brain slices were washed 3 1 h in PBS-T, then washed with TBS and mounted with fluorescent mounting medium (Component A, Molecular Probes, Eugene, Or) in secure-sealÔ spacers (Molecular Probes) attached to coverslips (No. 1½; 24 60 mm, Corning, NY) and sealed with coverglass No. 1 (Corning) and nail polish before being viewed with an Axiovert 200 epifluorescence microscope with an AxioCam MR monochrome camera (Carl Zeiss Inc.) [10, NA 0.30 Plan-Neofluar; 32, NA 0.40 Ph1 FL-D; 100, oil immersion, NA 1.3 Plan-Neofluar]. Lucifer Yellow was excited at 480/30, dichroic mirror 505DCLP, and emitter D535/40m. Alexa 568 was excited at D540/25, dichroic was 565 DCLP and emitted light was filtered through a bandpass filter D605/55m. AxioVision 3.1 software was used to collect data, and figures were assembled using Adobe Photoshop.
2.4. Statistics Parametric description of baseline membrane properties, such as those presented in Table 1, are described as mean absolute values with the 95% confidence interval (CI). Due to the variation in baseline membrane properties (i.e. IR, Cm, etc.) it was not possible to compare ethanol effects across cells. Thus, we employed a repeated measures design in which ethanol effects on membrane properties were compared within a given cell to the measurements made before ethanol exposure. Data from these experiments are presented as the mean percent difference relative to control with CI. The level of significance is presented in each figure. Statistical comparisons were with the Student’s t-test.
3. Results 3.1. Electrophysiological properties EtOH effects on astrocytes were evaluated in a total of 102 cells. The astrocytes showed variability in input resistance (range 5e180 MU), as well as in the apparent whole-cell capacitance values (range 1e297 pF). These measures are undoubtedly distorted by the presence of a gap junction-connected astrocyte syncytium. Astrocytes exhibited average resting membrane potentials (0 current levels) of 67 1.5 mV (n ¼ 64), which was close to the holding potential of 70 mV used in our voltage-clamp experiments. The cells were divided into two groups based on electrophysiological properties (Fig. 1). Of the analyzed cells, 87% exhibited a relatively linear I/V relationship and are referred to as passive astrocytes. Passive astrocytes exhibited a large time- and voltage independent conductance. The currents observed in the cells were almost certainly due to the combination of potassium and gap junction channel conductances, as almost no conductance was detected in cells loaded with CsCl in combination Table 1 Intrinsic properties of passive striatal astrocytes before and after 10 min exposure to 50 mM EtOH
IR (MU) Vm (mV) Cm (pF)
Control (n ¼ 15)
EtOH (% of control, n ¼ 15)
42 (CI 7.3) 64 (CI 3.0) 99 (CI 51)
125 (CI 13)*** 102 (CI 3.3)n.s. 72 (CI 17)*
***p < 0.001; *p < 0.05; n.s., Not Specified.
1101
with high [Ca2þ]i (Fig. 1). We did not observe any evidence of a fast inward current upon depolarization in passive astrocytes. Complex astrocytes expressed a combination of timeand voltage-activated current, as well as a large passive current (Fig. 1). The majority of the time- and voltage-dependent current was outward, but some cells exhibited fast inward currents upon depolarization. 3.2. EtOH effects on astrocytic ion currents Ethanol was applied to slices via bath perfusion and we measured responses to brief hyperpolarizing pulses (20 mV, 150 ms) delivered 1/10 s, and currents activated by de- and hyperpolarizing pulses (130 to 60) in 10 mV increments from the holding potential (70 mV). In passive astrocytes, EtOH reduced membrane conductance. This was most readily observed as a decrease in the amplitude of current activated by hyperpolarizing pulses (Fig. 2). Responses to these pulses stabilized following initiation of whole-cell recording, and subsequent application of 25 mM EtOH produced a decrease in current amplitude that developed gradually over the first 15 min of drug application. With continuous application of 25 mM EtOH we observed maximal inhibition of 27 5.5% after 40 min. According to chamber volume and flow rate, the buffer in the chamber would be completely exchanged within w1 min, and distribution of EtOH throughout the slice preparation should be fairly rapid given the amphipathic nature of this compound. Thus, the EtOH effects are delayed by a matter of a few minutes relative to the equilibration of alcohol in the slice. The amplitude of current produced by hyperpolarization did not return to pre-EtOH baseline levels following removal of alcohol from the preparation, even with perfusion times as long as 30 min (Fig. 2b). Cells not treated with EtOH exhibited a constant response to the hyperpolarizing current as long as the series resistance was stable (Fig. 2b). EtOH inhibited current generated by hyperpolarization and decreased slope conductance in passive astrocytes to a similar extent. The decrease in current amplitude was concentration dependent (Fig. 3), and 10 min exposure to 10, 25, 50 or 100 mM EtOH significantly reduced slope conductance in passive astrocytes to 88 6.0% (n ¼ 11), 83 6.7% (n ¼ 11), 79 5.9% (n ¼ 13) and 72 9.8% (n ¼ 10) of control values, respectively. Ion current in the presence of EtOH still lacked any time dependence, and retained the linear I/V relationship (Fig. 1). However, EtOH increased input resistance and decreased apparent cell capacitance (Table 1). Resting membrane current was not significantly affected by EtOH, which might be due to the fact that mean resting membrane potential is close to the holding potential (67 mV vs. 70 mV). After 10 min exposure to 10 mM, 25 mM, 50 mM or 100 mM EtOH resting membrane current in passive astrocytes was 100 4.0% (n ¼ 10), 96 5.5% (n ¼ 11), 96 6.2% (n ¼ 14) and 101 8.3% (n ¼ 9) of control. In complex astrocytes and neurons resting membrane current was 97 3.2% (n ¼ 12), and 94 6.6% (n ¼ 11) of control respectively, after 10 min exposure to 50 mM EtOH.
1102
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Fig. 1. Ion currents in passive and complex striatal astrocytes. Representative traces showing membrane current patterns and current/voltage relationship of one passive astrocyte (a), one complex astrocyte (b), and one passive cell in which intracellular KCl was replaced with CsCl þ 2 mM CaCl2 (c), before and after 10 min exposure to 50 mM EtOH. EtOH did not alter the lack of time-dependence or the linear shape of the I/V relationship, but reduced conductance, in passive astrocytes. Passive current was greatly reduced in cells filled with CsCl, and CaCl2 indicating that the major conductances in passive astrocytes are generated by potassium channels and gap junctions. EtOH had no effect on the remaining current in the CsCl þ CaCl2-filled cells. Membrane current patterns were measured in response to hyper- and depolarizing pulses given in 10 mV increments from a holding potential of 70 mV. I/V relationships plotted at the right were constructed from current measurements made at steady-state just before the end of the 250 ms voltage pulse.
In complex astrocytes, 10 min exposure to 50 mM EtOH did not affect current generated by hyperpolarization (96 6.3%, n ¼ 8) (Fig. 3). Likewise, 50 mM EtOH had no effect on currents generated by hyperpolarization in striatal neurons (102 7.3%, n ¼ 12) (Fig. 3). 3.3. Contribution of Kþ and gap junction conductances to the EtOH effect We examined the effects on the EtOH-induced inhibition of a number of manipulations designed to reduce particular cellular conductances. Blockage of potassium channels by substituting intracellular CsCl for KCl increased input resistance to 66 12 MU (n ¼ 10), decreased apparent capacitance (10 3.7 pF, n ¼ 10) but had no significant effect on membrane potential 57 5.6 mV (n ¼ 10). Substitution of KCl with CsCl in the patch pipette completely blocked the EtOH-mediated decrease in current amplitude (Fig. 4), and slope conductance (95 5.1%, n ¼ 11, 50 mM EtOH). Intracellular loading with 10 mM of the calcium chelator BAPTA
did not affect intrinsic properties in passive astrocytes. EtOH (50 mM) reduced hyperpolarization-induced current (Fig. 4), and decreased slope conductance by 23 5.4% under this condition (n ¼ 9), indicating that EtOH effects on astrocytes occur even in the presence of very low [Ca2þ]i. We attempted to block gap junction coupling via extracellular application of 1 mM octanol or 100 mM carbenoxolone, or intracellular loading of 2 mM CaCl2. Gap junctions were considered to be closed if input resistance markedly increased and apparent capacitance decreased within a 20 min time frame. None of the blockers used had a 100% success rate. Increased intracellular calcium produced changes indicative of gap junction closer in 64% of the cells examined. In cells where the blockers were effective input resistance increased (109 19 MU for octanol; 90 27 MU for carbenoxolone; 98 19 MU for intracellular CaCl2) and apparent capacitance decreased (25 12 pF; 22 12 pF; 15 6.6 pF for octanol, carbenoxolone, and intracellular CaCl2, respectively). Octanol and carbenoxolone shifted resting membrane potential to a more positive potential (43 7.9 mV and 46 12 mV,
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
1103
Fig. 2. Ethanol inhibition of current produced by hyperpolarization in passive astrocytes. (a) Currents generated by hyperpolarizing pulses of 20 mV (from a 70 mV holding potential) were allowed to stabilize following initiation of whole-cell recordings, and subsequent application of 25 mM EtOH produced a decrease in current amplitude that developed gradually over the first 15 min of drug application. Data are averaged values from seven passive astrocytes, and are presented as mean percent of control with 95% CI. (b) EtOH inhibition of current in response to hyperpolarization was largely irreversible in KCl filled astrocytes. The decrease in current amplitude mediated by 10 min exposure to 50 mM EtOH still remained after 30 min washout with EtOH-free solution. Current generated by hyperpolarization was stable in astrocytes not exposed to EtOH. Each measurement point is based on data from 15 cells, with 95% CI.
respectively), while 2 mM CaCl2 had no effect (61 8.3 mV). During blockage of gap junction coupling in cells treated with 1 mM octanol the inhibition by EtOH was intact (Fig. 4), and 50 mM EtOH reduced slope conductance to 83 11% of control values (n ¼ 10). Treatment with carbenoxolone or 2 mM CaCl2 did not eliminate the effects of EtOH, but the alcohol-induced decrease in the response to the hyperpolarizing pulse was significantly reduced (Fig. 4). Slope conductance was 88 5% (n ¼ 8) and 86 4% (n ¼ 13), respectively, of control values in the presence of 50 mM ethanol in these cells. We noted that when cells were treated with carbenoxolone or increased intracellular
CaCl2, the EtOH effect was fully reversible within 10 min upon washout of the alcohol (Fig. 5), whereas EtOH effects were largely irreversible under all of the other experimental conditions (with the exception of CsCl-filled cells where EtOH had no effect) (compare to Fig. 2). In those cells in which elevated intracellular Ca2þ had little effect on input resistance and capacitance EtOH effects were sustained during washout, indicating that the effect is connected to a reduction in gap junction coupling and not caused by the high [Ca2þ]i. EtOH did not affect conductance in cells loaded with CsCl and 2 mM CaCl2 (Fig. 1). Slope conductance was 100 3.5% of control, and current generated by hyperpolarization was 100 2.3% of control value (n ¼ 4). Spreading of Lucifer Yellow decreased by 46% in slices preincubated with 50 mM EtOH for 10 min before establishing whole cell recordings (Fig. 6). The number of stained cells was 507 287 (n ¼ 12) for control and 276 191 (n ¼ 8) in the EtOH treated slices ( p < 0.05). 3.4. Immunohistochemistry
Fig. 3. Ethanol inhibition is concentration dependent, and specific to passive astrocytes. Ten minutes exposure to 10, 25, 50 or 100 mM inhibited current generated by hyperpolarization in passive astrocytes in a concentration dependent manner, but did not affect hyperpolarization-induced current in complex astrocytes or neurons. Data are presented as percent of control with 95% CI. **p < 0.01, *p < 0.05.
Despite the use of high concentrations of Triton X-100 and long incubation times, antibody penetration into slices was poor. As patch clamped cells in most recordings were positioned deep within the slice, only a fraction (w20%) of the slices examined electrophysiologically were satisfactory stained. However, in the cases in which slices containing Lucifer Yellow stained cells were successfully labeled immunohistochemically the Lucifer-filled cells were positive for GFAP (n ¼ 25) (Fig. 7). Co-localization of Lucifer Yellow and OLIG1-, or MAP staining was not found indicating that none of the patch clamped cells were oligodendrocytes or neurons (based on 35 slices). No morphological differences between passive and complex astrocytes could be verified in the set of data presented here, mainly due to the fact that only a small number of complex cells were encountered, and many of these were not successfully labeled.
1104
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Fig. 4. (a) Graphs summarizing EtOH effects on the hyperpolarization-induced current during the indicated experimental manipulations. The EtOH effect was lost when CsCl was substituted for KCl in the internal solution, but remained in cells filled with 10 mM BAPTA, suggesting that ethanol affects calcium insensitive potassium channels. The EtOH effect was sustained during blockage of gap junctions with 1 mM octanol, but was significantly reduced when channels were closed with 100 mM carbenoxolone or intracellular loading with 2 mM CaCl2. Data are presented as mean percent of control values with 95% CI. #p < 0.05, ##p < 0.01, **p < 0.01, ***p < 0.001. (b) Representative traces showing currents generated by hyperpolarization in passive astrocytes before and during exposure to 50 mM EtOH. Astrocytes were held at 70 mV and subject to a hyperpolarizing pulse of 20 mV for 250 ms. Exposure to 1 mM octanol decreased the amplitude of the response in all cells, but EtOH reduced it further.
4. Discussion EtOH decreased capacitance, inhibited current in response to hyperpolarization and decreased slope conductance in passive astrocytes examined in rat striatal slices. The EtOH effect was blocked when KCl was replaced with CsCl in the internal solution, but not during chelation of intracellular calcium with BAPTA, suggesting that ethanol affects a calcium-insensitive potassium channel, most likely a passive potassium channel. EtOH did not affect currents generated by a hyperpolarizing pulse in complex astrocytes or neurons. The spread of Lucifer Yellow from single dye-filled cells revealed an extensively coupled astrocyte syncytium in the
striatum. Although the number of stained cells varied markedly from slice-to-slice, dye spreading was significantly reduced in brain slices pre-exposed to EtOH, indicating that EtOH acutely decreases gap junction dye permeability. Dye spreading in the striatum has previously been described between rat astrocytes (Hamon et al., 2002), while no dye spreading was reported between mouse striatal astrocytes (Rufer et al., 1996). However, the mouse astrocytes examined by Rufer et al. (1996) expressed connexin-43 and currents from surrounding cells could be detected, suggesting that there is a functional network. It is possible that the low temperature used in the Rufer et al. (1996) study might have contributed to the lack of dye spreading (Bukauskas and Weingart, 1993).
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Fig. 5. The decrease in current amplitude mediated by 10 min exposure to 50 mM EtOH was irreversible in KCl filled astrocytes but returned to baseline level within 10 min after beginning the washout of EtOH under conditions in which gap junction coupling was blocked by carbenoxolone or intracellular loading with 2 mM CaCl2. Graph describes current amplitude after 10 min exposure to 50 mM EtOH, and after 10 min rinsing with aCSF. Data are presented as mean percent of control values with 95% CI.
The increase in input resistance and decrease in capacitance detected during EtOH exposure might also be directly connected to a change in gap junction permeability. Apparent capacitance gives an indication of the membrane surface area of the cell and the decrease in this value could be a sign that the connection to other astrocytes has been reduced. Theoretically (Ohms law), an increase in input resistance would be expected when gap junctional currents are reduced. This has also been shown in rat slices from the hippocampus and striatum (Blomstrand et al., 2004; Hamon et al., 2002), while contradictory results were described for mouse astrocytes (Wallraff et al., 2004). However, in the latter study gap junctional blockers were only added for 3 min, and based on the thickness of the slices and the slow penetration of drugs into such slices it is possible that this duration of exposure
1105
is not sufficiently long to shut down enough gap junction channels to increase input resistance (Wallraff et al., 2004). All of the gap junction blockers that we employed in the present study produced an increase in input resistance, and thus this measure appears to be a reliable indicator of a decrease in gap junctional coupling. A decrease in dye spreading during EtOH exposure has previously been reported between rat astrocytes in primary astrocyte cultures (Adermark et al., 2004). The significant reduction in EtOH effect during blockage of gap junctions with carbenoxolone or increased [CaCl2]i gives a further indication that gap junction coupling is inhibited by EtOH. When we attempted to block gap junctions with octanol, the EtOH effect was not significantly reduced. The lack of effect of octanol could be explained by incomplete blockage of gap junction coupling, or could be due to nonspecific effects of octanol (Marcet et al., 2004; Swenson and Narahashi, 1980). Octanol treated cells showed a shift to a more positive resting membrane potential, and the variability in other membrane properties was larger in this treatment group in comparison to other treatments used. Thus, octanol appears to produce disturbances in cell physiology beyond those expected from simple closure of gap junctions. Carbenoxolone also produced a positive shift in resting membrane potential, but the series resistance in these experiments was more stable. Blockage of gap junctions with high intracellular calcium has been reported to be reversed during treatment of cells with high extracellular potassium (55 mM) or 0.4 mM kainate lasting for 10s of minutes (Enkvist and McCarthy, 1994). This effect was attributed to depolarization. However, depolarization was quite prolonged in this study, and both the high potassium and kainate treatments change the ionic balance across the cell membrane, as prolonged kainate would likely increase extracellular Kþ. Thus, it is not clear that the reversal of Ca2þ inhibition is solely due to depolarization. We did not observe any reversal of the effects of high intracellular Ca2þ in our experiments during brief depolarization to potentials of up to þ60 mV. The glial membrane properties were also quite stable in the presence of the high internal calcium. Thus, we believe that raising intracellular calcium is a reliable method for specifically eliminating gap junction coupling.
Fig. 6. The number of cells passively stained with Lucifer Yellow (0.05 mg/ml) was reduced by 46% in slices exposed to 50 mM EtOH for 10 min before establishing whole cell recordings. (a) Control slice; (b) stained cells in a slice exposed to 50 mM EtOH. Scale bar is 200 mm.
1106
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Fig. 7. Wide field images of cells immunoreactive for GFAP (a, c), co-labeled with Lucifer Yellow (b, d), showing that patch clamped cells were astrocytes. The concentration of Lucifer Yellow was low to prevent spreading to surrounding cells. We did not detect any Lucifer Yellow filled cells that stained positive for the oligodendrocyte marker OLIG1 or the neuronal marker MAP (based on evaluation of 35 brain slices). Scale bar is 20 mm.
EtOH effects on intrinsic membrane properties, current generated by hyperpolarization, and slope conductance were largely irreversible when gap junctions were open, but returned to baseline levels after washout of EtOH under two conditions that appeared to decrease gap junctional coupling, namely carbenoxolone or high intracellular calcium treatments. These findings suggest that EtOH affects gap junction coupling in a way that is sustained during EtOH withdrawal. Interestingly, Wentlandt and co-workers showed that the EtOH induced decrease in gap junction coupling between P19 cells did not recover after a 24-h withdrawal period (Wentlandt et al., 2004). The authors suggest that EtOH metabolism or secondmessenger activation after chronic EtOH exposure leads to functional closure of gap junctions. Our data indicate that the irreversible EtOH effect is induced within minutes. The complete blockade of EtOH effects in CsCl-filled cells is surprising. This manipulation should block potassium conductances, but would not necessarily alter the function of gap junctions. We did observe a decrease in apparent capacitance in the presence of intracellular CsCl indicating that this treatment alone might be sufficient to close gap junctions, and thus occlude both effects of EtOH. However, the conductance in Csþ loaded cells was still high, suggesting that gap junctions are not fully closed. Gap junctions formed by the connexin 43 protein (the major form present in astrocytes; Dermietzel et al., 1991) exhibit at least three different states; closed, open and residual (Valiunas et al., 1997), and single channel conductance
has been shown to depend on whether KCl or Cs-aspartate is present in the internal solution (Valiunas et al., 1997). Furthermore, it has been proposed that hemichannels formed by connexin 50 are regulated by monovalent cations (Srinivas et al., 2006), and this could be the mechanism through which internal Csþ also affects gap junction channels formed by connexin 43. In this context, it is interesting that the EtOH-mediated decrease in gap junction coupling in astrocyte cultures was blocked during manipulations that would affect potassium and sodium homeostasis (Adermark et al., 2004). It is possible that Csþ or the decrease in intracellular potassium affects the channel properties or gating in a way that interacts with the effect of EtOH, but it is also possible that the alcohol effect on gap junctions is somehow secondary to the change in potassium conductance. EtOH effects on passive potassium conductance and/or gap junction conductance could be mediated by a number of mechanisms. Ethanol-induced disturbances in intracellular signaling by PKC, and EtOH effects on the cytoskeleton, have been observed, and both of these processes are linked to the function of gap junction-forming connexins (Li et al., 2005; Newton and Messing, 2006; Shumilla et al., 2005; Zvalova et al., 2004). Future studies may elucidate the mechanisms underlying the EtOH effects. The data presented here suggest that EtOH effects on striatal astrocytes involve calcium insensitive potassium channels, and that EtOH modulates gap junction channels in a way that
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
is sustained during EtOH withdrawal. Astrocytes are important for maintaining potassium homeostasis, and effects of EtOH reported in the present study could produce secondary effects on neurotransmission and plasticity. Acknowledgments This work was sponsored by the Division of Intramural Clinical and Basic Research, NIAAA, NIH. We thank Dr. Lixian Dong for helpful discussions of gap junction pharmacology. References Adermark, L., Olsson, T., Hansson, E., 2004. Ethanol acutely decreases astroglial gap junction permeability in primary cultures from defined brain regions. Neurochem. Int. 45, 971e978. Allansson, L., Khatibi, S., Olsson, T., Hansson, E., 2001. Acute ethanol exposure induces [Ca2þ]i transients, cell swelling and transformation of actin cytoskeleton in astroglial primary cultures. J. Neurochem. 76, 472e479. Anderova, M., Antonova, T., Petrik, D., Neprasova, H., Chvatal, A., Sykova, E., 2004. Voltage-dependent potassium currents in hypertrophied rat astrocytes after a cortical stab wound. Glia 48, 311e326. Bekar, L.K., Loewen, M.E., Cao, K., Sun, X., Leis, J., Wang, R., Forsyth, G.W., Walz, W., 2005. Complex expression and localization of inactivating Kv channels in cultured hippocampal astrocytes. J. Neurophysiol. 93, 1699e1709. Blomstrand, F., Venance, L., Siren, A.L., Ezan, P., Hanse, E., Glowinski, J., Ehrenreich, H., Giaume, C., 2004. Endothelins regulate astrocyte gap junctions in rat hippocampal slices. Eur. J. Neurosci. 19, 1005e1015. Bordey, A., Sontheimer, H., 2000. Ion channel expression by astrocytes in situ: comparison of different CNS regions. Glia 30, 27e38. Braus, D.F., Wrase, J., Grusser, S., Hermann, D., Ruf, M., Flor, H., Mann, K., Heinz, A., 2001. Alcohol-associated stimuli activate the ventral striatum in abstinent alcoholics. J. Neural Transm. 108, 887e894. Bukauskas, F.F., Weingart, R., 1993. Temperature dependence of gap junction properties in neonatal rat heart cells. Pflugers Arch. 423, 133e139. Cotrina, M.L., Lin, J.H., Alves-Rodrigues, A., Liu, S., Li, J., AzmiGhadimi, H., Kang, J., Naus, C.C., Nedergaard, M., 1998. Connexins regulate calcium signaling by controlling ATP release. Proc. Natl. Acad. Sci. U.S.A. 95, 15735e15740. D’Ascenzo, M., Vairano, M., Andreassi, C., Navarra, P., Azzena, G.B., Grassi, C., 2004. Electrophysiological and molecular evidence of L(Cav1), N- (Cav2.2), and R- (Cav2.3) type Ca2þ channels in rat cortical astrocytes. Glia 45, 354e363. Dermietzel, R., Hertberg, E.L., Kessler, J.A., Spray, D.C., 1991. Gap junctions between cultured astrocytes: immunocytochemical, molecular, and electrophysiological analysis. J. Neurosci. 11, 1421e1432. Diamond, I., Gordon, A.S., 1997. Cellular and molecular neuroscience of alcoholism. Physiol. Rev. 77, 1e20. Enkvist, M.O., McCarthy, K.D., 1994. Astroglial gap junction communication is increased by treatment with either glutamate or high Kþ concentration. J. Neurochem. 62, 489e495. Fellin, T., Carmignoto, G., 2004. Neurone-to-astrocyte signalling in the brain represents a distinct multifunctional unit. J. Physiol. 559, 3e15. Fonseca, L.L., Alves, P.M., Carrondo, M.J., Santos, H., 2001. Effect of ethanol on the metabolism of primary astrocytes studied by (13)C- and (31)PNMR spectroscopy. J. Neurosci. Res. 66, 803e811. Freeman, M.R., 2005. Glial control of synaptogenesis. Cell 120, 292e293. Freeman, M.R., 2006. Sculpting the nervous system: glial control of neuronal development. Curr. Opin. Neurobiol. 16, 119e125. Gerdeman, G.L., Partridge, J.G., Lupica, C.R., Lovinger, D.M., 2003. It could be habit forming: drugs of abuse and striatal synaptic plasticity. Trends Neurosci. 26, 184e192.
1107
Giaume, C., Venance, L., 1998. Intercellular calcium signaling and gap junctional communication in astrocytes. Glia 24, 50e64. Grusser, S.M., Wrase, J., Klein, S., Hermann, D., Smolka, M.N., Ruf, M., Weber-Fahr, W., Flor, H., Mann, K., Braus, D.F., Heinz, A., 2004. Cueinduced activation of the striatum and medial prefrontal cortex is associated with subsequent relapse in abstinent alcoholics. Psychopharmacology (Berl) 175, 296e302. Haghighat, N., McCandless, D.W., Geraminejad, P., 1999. Effect of alcohol on energy storage of primary astrocytes and C6-glioma cells in vitro. Metab. Brain Dis. 14, 149e156. Hamon, B., Glowinski, J., Giaume, C., 2002. Intercellular diffusional coupling between glial cells in slices from the striatum. In: de Vellis, J. (Ed.), Neuroglia in the Aging Brain. Humana Press, Totowa, pp. 99e112. Haydon, P.G., 2000. Neuroglial networks: neurons and glia talk to each other. Curr. Biol. 10, R712eR714. Isokawa, M., McKhann, G.M., 2005. Electrophysiological and morphological characterization of dentate astrocytes in the hippocampus. J. Neurobiol. 65, 125e134. Janigro, D., Gasparini, S., D’Ambrosio, R., McKhann, G., DiFrancesco, D., 1997. Reduction of Kþ uptake in glia prevents long-term depression maintenance and causes epileptiform activity. J. Neurosci. 17, 2813e2824. Kronenberg, G., Wang, L.P., Synowitz, M., Gertz, K., Katchanov, J., Glass, R., Harms, C., Kempermann, G., Kettenmann, H., Endres, M., 2005. Nestin-expressing cells divide and adopt a complex electrophysiologic phenotype after transient brain ischemia. J. Cereb. Blood Flow Metab. 25, 1613e1624. Li, W., Hertzberg, E.L., Spray, D.C., 2005. Regulation of connexin43-protein binding in astrocytes in response to chemical ischemia/hypoxia. J. Biol. Chem. 280, 7941e7948. Lovinger, D.M., 1997. Alcohols and neurotransmitter gated ion channels: past, present and future. Naunyn Schmiedebergs Arch. Pharmacol. 356, 267e 282. Marcet, B., Becq, F., Norez, C., Delmas, P., Verrier, B., 2004. General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels. Br. J. Pharmacol. 141, 905e914. Modell, J.G., Mountz, J.M., Beresford, T.P., 1990. Basal ganglia/limbic striatal and thalamocortical involvement in craving and loss of control in alcoholism. J. Neuropsychiatry Clin. Neurosci. 2, 123e144. Nedergaard, M., 1994. Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science 263, 1768e1771. Nedergaard, M., Ransom, B., Goldman, S.A., 2003. New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci. 26, 523e530. Newton, P.M., Messing, R.O., 2006. Intracellular signaling pathways that regulate behavioral responses to ethanol. Pharmacol. Ther. 109, 227e237. Othman, T., Sinclair, C.J., Haughey, N., Geiger, J.D., Parkinson, F.E., 2002. Ethanol alters glutamate but not adenosine uptake in rat astrocytes: evidence for protein kinase C involvement. Neurochem. Res. 27, 289e296. Parkerson, K.A., Sontheimer, H., 2004. Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes. Glia 46, 419e436. Rufer, M., Wirth, S.B., Hofer, A., Dermietzel, R., Pastor, A., Kettenmann, H., Unsicker, K., 1996. Regulation of connexin-43, GFAP, and FGF-2 is not accompanied by changes in astroglial coupling in MPTP-lesioned, FGF2-treated parkinsonian mice. J. Neurosci. Res. 46, 606e617. Shumilla, J.A., Liron, T., Mochly-Rosen, D., Kendig, J.J., Sweitzer, S.M., 2005. Ethanol withdrawal-associated allodynia and hyperalgesia: agedependent regulation by protein kinase C epsilon and gamma isoenzymes. J. Pain 6, 535e549. Slezak, M., Pfrieger, F.W., 2003. New roles for astrocytes: regulation of CNS synaptogenesis. Trends Neurosci. 26, 531e535. Srinivas, M., Calderon, D.P., Kronengold, J., Verselis, V.K., 2006. Regulation of connexin hemichannels by monovalent cations. J. Gen. Physiol. 127, 67e75. Swenson, R.P., Narahashi, T., 1980. Block of sodium conductance by n-octanol in crayfish giant axons. Biochim. Biophys. Acta. 603, 228e236.
1108
L. Adermark, D.M. Lovinger / Neuropharmacology 51 (2006) 1099e1108
Valiunas, V., Bukauskas, F.F., Weingart, R., 1997. Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells. Circ. Res. 80, 708e719. Wallraff, A., Odermatt, B., Willecke, K., Steinhauser, C., 2004. Distinct types of astroglial cells in the hippocampus differ in gap junction coupling. Glia 48, 36e43. Walz, W., 2000. Controversy surrounding the existence of discrete functional classes of astrocytes in adult gray matter. Glia 31, 95e103.
Wentlandt, K., Kushnir, M., Naus, C.C., Carlen, P.L., 2004. Ethanol inhibits gap-junctional coupling between P19 cells. Alcohol Clin. Exp. Res. 28, 1284e1290. Zhou, M., Schools, G.P., Kimelberg, H.K., 2006. Development of GLAST(þ) astrocytes and NG2(þ) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive. J. Neurophysiol. 95, 134e143. Zvalova, D., Cordier, J., Mesnil, M., Junier, M.P., Chneiweiss, H., 2004. p38/ SAPK2 controls gap junction closure in astrocytes. Glia 46, 323e333.