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The Journal of Neuroscience, September 22, 2004, 24(38):8322-8332; doi:10.1523/JNEUROSCI.1536-04.2004
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Cellular/Molecular
Alcohol Tolerance in Large-Conductance, Calcium-Activated Potassium Channels of CNS Terminals Is Intrinsic and Includes Two Components: Decreased Ethanol Potentiation and Decreased Channel Density
Andrzej Z. Pietrzykowski,1 Gilles E. Martin,1 Sylvie I. Puig,1 Thomas K. Knott,2 Jose R. Lemos,2 andSteven N. Treistman1 Departments of 1Neurobiology and 2Physiology, University of Massachusetts School of Medicine, Worcester, Massachusetts 01605
Abstract
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Abstract
Introduction
Tolerance is an important element of drug addiction and provides a model for understanding neuronal plasticity. The hypothalamic–neurohypophysial system (HNS) is an established preparation in which to study the actions of alcohol. Acute application of alcohol to the rat neurohypophysis potentiates large-conductance calcium-sensitive potassium channels (BK), contributing to inhibition of hormone secretion. A cultured HNS explant from adult rat was used to explore the molecular mechanisms of BK tolerance after prolonged alcohol exposure. Ethanol tolerance was intrinsic to the HNS and consisted of: (1) decreased BK potentiation by ethanol, complete within 12 min of exposure, and (2) decreased current density, which was not complete until 24 hr after exposure, indicating that the two components of tolerance represent distinct processes. Single-channel properties were not affected by chronic exposure, suggesting that decreased current density resulted from downregulation of functional channels in the membrane. Indeed, we observed decreased immunolabeling against the BK-subunit on the surface of tolerant terminals. Analysis using confocal microscopy revealed a reduction of BK channel clustering, likely associated with the internalization of the channel.
Key words: addiction; alcohol; ethanol; explant; tolerance; BK channel; terminals
Introduction
Tolerance represents an important component of drug addiction. Various forms of tolerance (e.g., acute, chronic) are defined by their time course (Kalant, 1998). The hypothalamic–neurohypophysial system (HNS), responsible for the release of the peptide hormones vasopressin (AVP) and oxytocin (OXT), has proven to be a useful preparation to study the acute and chronic actions of alcohol, including tolerance. The effects of ethanol on channel function in this system have important physiological and behavioral consequences, resulting from the wide-ranging central and peripheral actions of AVP and OXT (Lemos, 2001
Although significant progress toward understanding the molecular mechanisms of tolerance in the HNS has been made, the use of the whole animal has been limiting. First, studies of the chronology of tolerance, using the sucrose fade technique, are impeded by the period necessary for the establishment of consistent blood alcohol levels (Knott et al., 2000
We demonstrate two distinct components of intrinsic tolerance: decreased potentiation by alcohol evident within 12 min of exposure, and a decrease in current density that develops over a 24 hr period. Single-channel recordings indicate that conductance of the channel is not changed in the tolerant terminals, suggesting that the decrease in current density corresponds to a diminished number of channels in the membrane. This is corroborated by immunohistochemical studies showing decreased channel labeling on the surface of tolerant terminals. Moreover, quantitative analysis revealed a change in the expression pattern from cluster-type in naive terminals to a diffuse pattern in tolerant terminals.
Preliminary data were presented in an abstract form (Pietrzykowski et al., 2001
Materials and Methods
Explant preparation and culture
Male Sprague Dawley adult rats (3–5 weeks old) were obtained from Taconic Farms (Germantown, NY). All procedures were performed in accordance with the National Institutes of Health guidelines on the Care and Use of Animals and study protocol approved by the University of Massachusetts Animal Care and Use Committee. After decapitation, the brain and pituitary with intact pituitary stalk were removed very rapidly from the skull and chilled in oxygenated dissection buffer (in mM): 120 NaCl, 5 KCl, 1.0 MgCl2, 1.9 CaCl2, 2.0 EGTA, 10 HEPES, 5.0 NaHCO3, 15.0 glucose, 0.5 glutamine (Glutamax; Invitrogen, Gaithersburg, MD), and 100 U/ml to 0.1 mg/ml penicillin–streptomycin (Invitrogen), pH 7.2, 310 mOsm. To increase cell survival, dissection buffer contained: (1) low levels of free calcium (3 µM), (2) B27 supplement with antioxidants (1:50; Invitrogen), and (3) 1 mM nitro-arginine and kynurenic acid. Under a dissection microscope, the anterior pituitary was removed, and a hexagonal block of tissue was carved out (by cutting rostral to the optic chiasm, parallel to the optic tract, lateral to the median eminence and caudal to the stalk, and undercutting at a depth of 1–2 mm). Next, tissue was trimmed to the final thickness of 550–650 µm to enable nutrients and oxygen penetration despite the lack of vascular perfusion (Fujii et al., 1982Ethanol treatment
A constant ethanol concentration was maintained by placing an open dish with 20 mM ethanol near the dish containing the ethanol-treated explant, in a closed, but not sealed container, which allowed exchange of gases. Ethanol levels measured in both the media and the tissue, using an NAD-ADH kit (Sigma) according to manufacturer's instructions, indicated a constant level of 20 mM ethanol at all exposure times tested (0.5, 1, 3, 6 and 24 hr). For exposures <30 min, terminals were dispersed first and then exposed to ethanol during electrophysiological recording.Viability assay
Immunostaining. Explants were fixed in 4% paraformaldehyde (PFA). After immersion in 20% sucrose, explants were embedded in 6% gelatin–egg yolk mixture in cardboard molds, and exposed to concentrated formaldehyde vapors for at least 3 d (4°C). After hardening, 60 µm sections were cut on a freezing microtome, and sections were placed in Net-well carriers. The floating sections were permeabilized and blocked and incubated alternately with the primary monoclonal mouse antibodies: G38 (against AVP-neurophysin) or G41 (anti-OXT–neurophysin) (1:300; a gift from H. Gainer, Laboratory of Neurochemistry, National Institute on Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD) (Ben Barak et al., 1985Unbiased stereological determination of magnocellular neuron viability. An unbiased stereological technique (West et al., 1991
40 µm diameter) filled with hormone, with long processes and a large nonpyknotic nucleus. Magnocellular neurons with shrunken cytoplasm and/or pyknotic nuclei were considered apoptotic (Vutskits et al., 1998
NH terminal preparation
Neurohypophysial terminals were homogenized as previously described (Cazalis et al., 1987Electrophysiology
Dissociated terminals were identified by their spherical shape, size (2–8 µm in diameter), lack of nuclei, and golden color under Hoffmann–phase contrast optics. First they were bathed in ethanol-free low-calcium (3 µM) Locke's solution (in mM: 142 NaCl, 2.0 KCl, 2.0 CaCl2, 2.0 MgCl2, 13.0 glucose, and 15.0 HEPES, pH 7.3, 305 mOsm), which subsequently was changed into ethanol-free normal calcium Locke's solution (2.2 mM CaCl2, no EGTA). Average time before ethanol challenge was 60 min.Whole-cell recordings
Electrodes were pulled from thin-walled borosilicate glass (Drummond Scientific, Broomall, PA) on a vertical puller (David Kopf Instruments, Tujunga, CA) and fire-polished (Narashige, Tokyo, Japan) to give a final resistance of 5–10 M(2–4 M
Single-channel recordings
Unitary current recordings were obtained from excised, inside-out patches using standard patch-clamp techniques. Currents were recorded in symmetrical 135 mM K+ using a patch-clamp amplifier (Axopatch 200; Axon Instruments) at a sampling rate of 5 kHz and filtered at 1 kHz using an eight pole Bessel filter. Data were acquired and analyzed using pClamp 6.02 software.Superfusion and drug application
Terminals in whole-cell configuration or patches excised from the membrane were exposed to 5 µM [Ca2+]free control solution flowing from a micropipette (1 mm diameter; WPI, Sarasota, FL), and the potential across the membrane was set to produce a relatively low open probability (NPoIn the perforated patch configuration, BK current was isolated by adding 4-aminopyridine to the bath (7 mM final concentration) to block the fast inactivating (IA) potassium current (Thorn et al., 1991
Data analysis
NPo was determined in the presence of 10 µM Ca2+ and was used as an index of channel activity. NPo values were calculated from all-points amplitude histograms by fitting the histogram with a sum of Gaussian functions using a Levenberg–Marquardt algorithm. NPo data as a function of voltage were fitted with a Boltzmann function of the type: Po = (1 + exp – K(V – V0.5))–1, where K is the logarithmic potential sensitivity and V0.5 the potential at which Po is half-maximal. When the NPo–voltage relationship is fitted by a Boltzmann curve, a plot of lnNPo versus voltage is linear at low values of Po (Dopico et al., 1996The relationship between [Ca2+]i and the channel steady-state Po is usually a sigmoid function that can be fitted according to a Hill equation: Po = Pmax[Ca2+]inH/(K + [Ca2+]inH)where Po is the steady-state open probability, Pmax is the maximum open probability, nH is the slope factor (or Hill coefficient), and the nth root of K gives an estimate of the midpoint of the activation curve and the sensitivity of the channel to [Ca2+]i. The slope factor provides a minimum estimate of the number of Ca2+ ions involved in maximally activating the channel (McManus, 1991
Duration of open times was measured with half-amplitude threshold analysis. Mean open time was calculated based on an equation: To = NPoT/Xo for multiple channel recordings [N = number of channels; Po = open probability; T = time of observation (in milliseconds); Xo = number of openings]. A maximum-likelihood minimization routine was used to fit curves to the distribution of open times. Determination of the minimum number of terms for adequate fit was established using a standard F statistic table (significance level, p < 0.01). The unitary conductance (
) was taken as the slope of the unitary current amplitude–voltage relationship. Values for unitary current were obtained from the Gaussian fit of all-points amplitude histograms by measuring the distance between the modes corresponding to the closed state and the first opening level. For all experiments, voltages given correspond to the potential at the intracellular side of the membrane.
Immunocytochemistry, confocal analysis, and quantification of BK expression
Terminals were isolated as described above, fixed in 2% PFA, and double-labeled using mouse antibodies against AVP (or OXT) and rabbit antibodies against BK channelStatistics
Unless otherwise indicated, statistical significance between the various treatment groups was analyzed using Student's t test (Statistica, version 5.5; StatSoft, Tulsa, OK). All data are expressed as mean ± SEM, and p values <0.05 were considered to be statistically significant.
Results
HNS explants contain viable AVP and OXT magnocellular neurons with intact axons
Because of the nonplanar route of axons from the SON to the NH (Silverman and Zimmerman, 1983
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Figure 1. HNS explant contains viable AVP and OXT neurons of the intact SON–NH. A, Bright-field top view of an HNS explant. B, Diagram of HNS explant showing position of magnocellular neuron compartments: cell bodies, axons, and terminals. C, Immunofluorescent image (above) and sagittal diagram (below) of the SON–NH tract (AVP-positive, similar OXT-positive results not shown) indicate that axonal projections from SON magnocellular neurons to the neurohypophysis were preserved, and the SON–NH tract is the only structure present in the HNS explant. Note the winding path of axons from the SON, above the optic tract, below the sole of the third ventricle, through the median eminence to the neurohypophysis. D, Magnocellular neurons in SON were counted to estimate the viability of the HNS explants. Representative immunofluorescent grayscale images (top) of AVP neuron in the SON from explants cultured in 20 mM ethanol for 24 hr (similar data for OXT and fresh and naive explants not shown). Note the large cell body with long processes filled with hormone (left image) and large, nonapoptotic nucleus with diffuse Hoechst staining (right image, arrow).Other neurons are not visible because of the irout-of-focus location. The exact number of neurons in SON was estimated using stereological techniques (see Material and Methods) and is shown in the graph (bottom). Cap on each bar represents SD. ME, Median eminence; MM, medial mammilliary nucleus; PVN, paraventricular nucleus; 3V, third ventricle. Scale bars: A, 1.5 mm; C, 430 µm; D, 20 µm.
Chronic, low-dose ethanol treatment does not influence magnocellular neuron viability
High doses of ethanol (30 min, ethanol penetrated the tissue of the HNS explant and was maintained at a constant 20 mM level throughout the treatment. Viability of magnocellular neurons was unaffected by 48 hr culture with or without 24 hr ethanol treatment (Fig. 1D). Viable AVP and OXT magnocellular neurons have large cell bodies and processes filled with hormone. Using Hoechst dye, we observed viable cells, in which the large, weakly stained nuclei with loosely coiled chromatin suggested the absence of apoptosis (Bonde et al., 2002
BK channels retain normal properties in cultured HNS explant
Figure 2A shows typical single-channel recordings from an inside-out patch from a BK channel in a NH terminal from a naive explant. We tested the voltage dependence of this channel by exposing its intracellular face to 5 µM free Ca2+ and changing the holding potential. At –60 mV, the BK channel remained mostly in a closed state and showed only brief openings. As more depolarized potentials (–40 and –20 mV) were reached, the time spent in the open state increased (Fig. 2A). We also tested the calcium dependence of BK channels by holding the membrane potential at +30 mV and exposing the cytoplasmic face of the channel to increasing Ca2+ concentrations (Fig. 2B). In the presence of 1 µM Ca2+, the BK channel showed only sporadic openings. After switching to 5 µM Ca2+, channel activity increased, to finally reach near maximum open probability in 10 µM Ca2+. Finally, a unitary conductance of 215 pS, a value typical of BK channels, was observed in symmetrical 135 mM K+ (Fig. 2C). Thus, BK channel basic properties (Wang et al., 1992
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Figure 2. Voltage and calcium dependence of BK channels in naive NH terminals. A, Representative currents from a single BK channel, recorded in an inside-out patch at different potentials in the presence of 10µM free [Ca2+]i. Channel activity increases as the patch membrane is held at more depolarized potentials. B, Activity of a single BK channel as in A recorded at +30 mV in the presence of various [Ca2+]i. The activity increases as the cytosolic face of the channel is exposed to increasing concentrations of calcium. C and O1, adjacent to the traces, indicate the closed and the open states, respectively. C, Unitary conductance was determined by fitting the I–V relationship by linear regression.D, Ethanol-mediated potentiation of BK current is voltage-independent. Graph shows the average BK current amplitude (n = 5) as a function of the membrane voltage measured in whole-cell voltage clamp. Each terminal served as its own control. A 50 mM concentration of ethanol increased BK current amplitude at all the potentials tested between –10 and +70 mV in a voltage-independent manner (inset). Representative traces show BK currents recorded from a naive terminal and evoked at different membrane potentials (20 mV steps) before (control) and after 50 mM ethanol application. The fast transient current at the beginning of the voltage pulse is caused by sodium channels (TTX was omitted from the bath solution).
BK channel tolerance to ethanol in terminals is intrinsic
Next, we examined whether 24 hr chronic ethanol treatment of the explant produces changes in BK channel properties similar to those observed in terminals obtained from animals on an ethanol diet (Knott et al., 2002
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Figure 3. BK channels exhibit intrinsic tolerance to ethanol after 24 hr. A, Effects of acute ethanol in freshly isolated, naive, and chronic explants. Inside-out terminal patches were exposed to 5 µM free-Ca2+ control solution, and the potential across the membrane was set to produce low open probability (NPo around 0.2), to allow observation of ethanol (100 mM) potentiation. Whereas potentiation is evident in freshly isolated and naive terminals, chronic terminals show lack of ethanol effect on BK activity as shown in representative traces at the left of each panel. All-points amplitude histograms summarizing these effects are shown on the right.The washout histogram is omitted from the graph for clarity. Differences in unitary current amplitude reflect different holding potentials that were –50, –50, and –30 mV for patches from fresh, naive, and chronic conditions, respectively. B, The increase of BK channel open probability by acute ethanol challenge in naive (n = 7) and chronic (n = 12) explants. BK channel activity is expressed as the ratio of BK channel open probability after acute ethanol challenge over its activity before drug application (NPo EtOH /NPo Control), where 1 = no potentiation. Chronic ethanol treatment decreases BK channel potentiation by ethanol (p < 0.05) compared with the naive group. C, BK currents in terminals were recorded in whole-cell patch clamp to determine current densities. Membrane potential was held at –70 mV and depolarized for 500 msec from –50 to +90 mV in 20 mV increments. D, Average current densities (±SEM) recorded in naive (n = 5) and chronic (n = 4) terminals were significantly smaller (
In addition to the decreased potentiation by acute drug challenge, BK current densities obtained in terminals from explants exposed to ethanol for 24 hr were significantly lower than in terminals from naive explants. In terminals from naive explants, depolarizing the membrane to +50 mV (from –70 mV) evoked a strong BK current (196 ± 43 pA). In contrast, after 24 hr ethanol treatment, a similar voltage step elicited only 56 ± 36 pA of BK current (Fig. 3C). When current was expressed as a function of membrane surface area (determined by membrane capacitance), we found a dramatic decrease in current density after chronic ethanol treatment, from 14.2 ± 3 pA/10 µm2 (n = 5) in naive explants to 2.71 ± 2 pA/10 µm2 (n = 4) in chronic explants (p < 0.05) (Fig. 3D).Thus, chronic tolerance to ethanol observed in neurohypophysial terminals from cultured explants was intrinsic and did not require the actions of elements originating outside of the HNS.
Time course of tolerance suggests two distinct processes
We next determined the time course, beginning at 30 min, of the two components of tolerance evident at 24 hr. Macroscopic currents in terminals from HNS explants cultured for various periods of time in ethanol were obtained, using whole-cell patch clamp recordings. Two distinct time courses were observed. A rapid reduction in BK ethanol potentiation was evident within 30 min of drug exposure and was sustained for the 24 hr test period, whereas BK current density responded more slowly to the ethanol treatment, showing partial reduction in current density after 6 hr of ethanol exposure, and reaching a near-minimal value after 24 hr (Fig. 4A). The finding that decreased potentiation was already fully present at the 30 min time point led us to examine the development of this form of tolerance during the first few minutes of ethanol exposure. Because short-latency changes might be complicated by a time lag attributable to ethanol penetration into the explant, we altered the protocol, to examine the reduction of ethanol potentiation of BK current during continued exposure to ethanol using the perforated patch clamp technique of an isolated terminal from an ethanol-naive rat. It is evident (Fig. 4B) that potentiation is decreasing within 4 min of drug exposure, and this form of tolerance is fully developed by 12 min of exposure. Introduction of an incremental 50 mM ethanol (final concentration, 100 mM) at the 13 min time point did not result in the reinitiation of potentiation (data not shown). Representative traces are shown in Figure 4C.
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Figure 4. Time course of the development of ethanol tolerance of BK channels. A, HNS explants were cultured on Millipore inserts and exposed to ethanol for 0, 0.5, 1, 6, and 24 hr (n = 4–10). Ethanol tolerance of BK channels in the cultured NH terminals is manifested as both decreased sensitivity to ethanol, evident within 30 min of drug exposure and sustained for the entire 24 hr observation period, and reduced BK current density, evident after 6 hr, and continuing to decrease during the 24 hr observation period. B, The time course of decreased potentiation of BK current during the first few minutes was determined using the perforated patch-clamp technique with an isolated terminal from an ethanol-naive neurohypophysis (n= 2). The graph demonstrates the rapid loss of potentiation in the continued presence of ethanol. C, Representative traces of BK current from specified time points in B.
Single-channel properties of BK remain unchanged after chronic ethanol treatment
The observed decrease in current density could result from either altered individual channel properties or from a downregulation of functional channels (or both). We tested the first possibility by examining gating properties, kinetics, and conductance of terminal BK channels from naive and chronic (24 hr ethanol exposure) explants.Voltage and calcium activation
Figure 5A shows that the open probability of BK channels obtained from naive (open markers) and chronic (filled markers) terminals, as a function of calcium concentrations and voltage, does not differ. In both groups, for each [Ca2+]i the NPo–voltage relationship could be well fitted with a sigmoid function (see Materials and Methods). Figure 5B shows the data plotted in a manner that illustrates the lack of effect of chronic treatment on BK channel voltage sensitivity. The reciprocal of the slope of the ln(Po)–V relationship at low Po in the presence of 10 µM free-Ca2+ indicated that the voltage necessary to produce an e-fold change in open probability was similar in naive (28 ± 1.22 mV) and chronic terminals (29 ± 1.21 mV, see Materials and Methods).
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Figure 5. Chronic ethanol does not alter BK channel characteristics. A, Normalized NPo as a function of voltage at three different [Ca2+]i in naive (open symbols) and chronic (filled symbols) BK channels were fitted with a Boltzmann relationship of the type: Po = (1 + exp – K(V – V0.5)) –1, where K is the logarithmic potential sensitivity, and V0.5 the potential at which Po is half-maximal. At each calcium concentration, naive and chronic BK channels are very similar. The Po–V curve is shifted similarly along the voltage axis to more negative potentials as [Ca2+]i is increased. B, Data plotted in a manner that illustrates the lack of effect of chronic treatment on BK channel voltage sensitivity. The reciprocal of the slope of the ln(Po)–V relationship at low Po in the presence of 10µM free-Ca2+ indicated that the voltage necessary to produce an e-fold change in open probability was similar in naive (28 ± 1.22 mV) and chronic terminals (29 ± 1.21 mV). C, V0.5 values, obtained from Boltzmann fits displayed in A (dashed line), are now plotted as a function of [Ca2+]i on a logarithmic scale. Data points are fitted by a linear regression (r = 0.99). The leftward shift inV0.5 as [Ca2+]i increases is very similar in naive and chronic BK channels. D, The averaged mean open time of BK channels from naive (n = 6) and chronic explants (n = 7) are not different. Mean open time was calculated based on the equation: To = NPoT/Xo for multiple channel recordings [N, Number of channels; Po, open probability; T, time of observation (in milliseconds), Xo, number of openings]. E, The mean BK channel conductance from naive (n = 12) and chronic (n = 11) terminals obtained by fitting I–V plots (Fig. 2C) are not different.
We compared the calcium sensitivity of BK channels from naive and chronic explants. This is illustrated by the plot of V0.5 (in millivolts) as a function of free-Ca2+, in which V0.5 decreases as [Ca2+]i increases (Fig. 5C). We observed no difference between the naive and the chronic group. We also calculated the Hill coefficient by fitting the slope of the relationship between NPo and [Ca2+]i and found it was similar in naive (1.15) and chronic explants (1.07; plot not shown).Kinetics
The mean open time of BK channels in the naive group was 3.5 ± 0.4 msec (n = 6), which did not differ from that of BK channels from the chronic group (3.44 ± 0.55 msec; n = 7) (Fig. 5D).Conductance
Single-channel conductance was also unaffected by chronic exposure. In the naive group, BK channels had a conductance of 213 ± 3.37 pS (n = 11), which is in accordance with previous reports of BK channels isolated from NH (Dopico et al., 1996Chronic ethanol causes declustering and redistribution of BK channels
The finding that single-channel properties were unaltered after the development of tolerance suggested that the decrease in current density might result from a decrease in BK channel expression in the terminal membrane. To address this question, we measured expression of BK channel protein in terminals, using a specific antibody directed against the main (pore-forming)
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Figure 6. Chronic ethanol changes distribution of BK channels in neuronal terminals. A, Representative immunofluorescent confocal images (in a grayscale) of naive and chronic HNS terminals stained for BK pore-forming
In addition to BK channel redistribution, we also found that chronic ethanol disturbed the expression pattern of BK channels in the terminal membrane. In terminals obtained from naive explants, BK channels were present mainly in clusters, whereas in the membrane of the terminals from chronic explants, clusters were rarely seen, and BK expression was more diffuse. Quantitative analysis showedthat in naive terminals, BK clustersaveraged 6.5 per 100 µm2 of terminal membrane (Fig. 7A), whereas in terminals isolated from chronic explants, clusters were observed in less than half of the terminals (43%), and when present, averaged only 1 per 100 µm2 (Fig. 7A) (p < 0.003). In the remaining 57% of chronic terminals, BK channels (although present in the membrane) were not clustered. Thus, chronic ethanol caused declustering of BK channels in the membrane of neuronal terminals.
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Figure 7. Chronic ethanol causes declustering of BK channels and reduces density of BK channels in clusters. Confocal images from naive and chronic terminals were used in this study. The number of BK clusters, their size, and the intensity profiles were determined using Leica quantification software (version 2.00; Microsystems). A, Quantification of the number of clusters in naive (n = 12) and chronic (n = 14) terminals shows a significant decrease after chronic ethanol treatment. B, Representative images of a naive and a chronic cluster showing axes used to determine three-dimensional density of BK in clusters. C, Intensity profiles of BK clusters in naive and chronic terminals measured along X, Y, and Z axes as shown in B. D, Quantification of BK density in naive (n = 40) and chronic (n = 8) clusters indicates that chronic ethanol causes an approximately twofold decrease in the BK density per cluster. (*p < 0.05).
Chronic ethanol decreases BK channel density in clusters
Next, we determined whether BK channel density in clusters was altered after chronic ethanol treatment. Using confocal sections (in which clusters appeared oval-shaped), the dimensions of each cluster along the X, Y, and Z axes (Fig. 7B), and the intensity of BK channel expression in each section were determined. The dimensions of clusters in naive terminals (n = 40) (X, Y, and Z measurements) were 367 ± 82, 261 ± 56, and 843 ± 175 nm, respectively, and did not differ significantly from the corresponding values obtained from ethanol-treated terminals (n = 8) (397 ± 55, 234 ± 52, and 834 ± 38 nm). However, although the size of clusters remained the same, the intensity of BK signal in each cluster was decreased along all axes after chronic ethanol treatment (Fig. 7C). Figure 7D illustrates that 24 hr chronic ethanol treatment of HNS explants caused a significant (p < 0.05) decrease in BK channel density in clusters in neuronal terminals.
Discussion
The present study examines the effects of ethanol on BK channel expression and function in CNS terminals from cultured HNS explants. The HNS, through the release of the peptide hormones AVP and OXT, plays a critical role in physiology and behavior. In addition to its physiological role in the circulatory system and urinary tract (Lemos, 2001Earlier findings indicated that the acute actions of ethanol differ between cellular compartments of magnocellular neurons, with BK channels located in the terminals potentiated by ethanol, whereas BK channels in the cell bodies were insensitive to ethanol (Dopico et al., 1999b
Here, we first demonstrate the successful preparation of a viable HNS explant, harvested from the adult rat brain, which can be cultured for a time suitable to study chronic ethanol effects. HNS explants, like organotypic cultures, have many advantages over cell cultures, including preservation of the three-dimensional architecture found in vivo (Sladek and Knigge, 1977
An important finding in this study was the identification of the intrinsic character of tolerance to ethanol in BK channels of neuronal terminals. The explants did not contain other brain structures [e.g., organum vasculosum laminas terminalis (OVLT); suprachiasmatic nucleus (SCN)], which could modulate the function of magnocellular neurons. The actions of elements originating outside of the brain were also eliminated. Thus, molecular mechanisms of tolerance are contained within the HNS.
Ethanol tolerance observed in the cultured explant and manifested as both diminished potentiation and decreased current density, qualitatively resembled the changes observed in terminals obtained from animals chronically exposed to the drug for 3 weeks. However, the explant allowed us to establish that these two forms of tolerance represent distinct processes. The reduced potentiation was very rapid and evident within a few minutes of exposure, whereas the reduced current density did not near completion until 24 hr of exposure. Neither form of tolerance required the 3 week exposure period that was necessary to obtain stable blood alcohol levels in the ingesting animal (Knott et al., 2000
C-fos in encoding long-lasting nervous system changes after drug exposure (Nestler, 2002
A combination of immunohistochemistry and single-channel recording indicated that the decreased BK current density resulted from a reduced number of membrane channels, rather than from a change in single-channel properties (see model in Fig. 8). Alteration of the number of channels expressed in the terminal membrane serves as an important mechanism regulating synaptic efficacy and neurotransmitter release (Man et al., 2000
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Figure 8. Summary of modulation of BK channel function and distribution in neuronal terminals by chronic ethanol. After 24 hr exposure, BK channels are (1) less potentiated by acute challenge, (2) more internalized, (3) less clustered in the membrane, and (4) less dense within remaining clusters.
Chronic ethanol treatment resulted in the declustering and lateral diffusion of BK channels in the terminal membrane (Fig. 8). Clustering of another potassium channel, Kv1.4, is essential for suppressing internalization and degradation of that channel and serves as a mechanism for the regulation of channel density in the plasma membrane (Jugloff et al., 2000Thus, the development of BK channel tolerance to ethanol consists of two distinct components: a rapid decrease of potentiation by ethanol of individual channels and a slower process of diminishing channel density on the CNS terminal surface. The cultured HNS explant offers a powerful preparation in which to describe and study the mechanisms underlying tolerance in CNS terminals.
Footnotes
Received April 22, 2004; revised July 23, 2004; accepted August 11, 2004.This work was supported by National Institutes of Health Grant AA08003 (S.N.T.). We thank Paula Feinberg-Zadek, Noreen Rossi, and Hector Marrero for helpful comments on the explant preparation, Harold Gainer for G38 and G41 antibodies, Thomas Schoenfeld for his assistance and use of stereological equipment, Robert O'Connell and David Stevens for assistance in statistical analysis, and members of Jose Lemos' and our research groups for insightful discussions.
Correspondence should be addressed to Steven Treistman, Department of Neurobiology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605. E-mail: steven.treistman{at}umassmed.edu.
DOI:10.1523/JNEUROSCI.1536-04.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/248322-11$15.00/0
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