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Ca²⁺ Flux and Signaling Implications by Nicotinic Acetylcholine Receptors in Rat Medial Habenula

¹Department of Neurobiology, McKnight Brain Institute, ²Civitan International Research Center, and the ³Vision Science Graduate Program, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 4 October 2005; accepted in final form 11 October 2006

	ABSTRACT

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The fraction of inward current carried by Ca²⁺ (FCa²⁺) through nicotinic acetylcholine receptors (nAChRs) on acutely isolated rat medial habenula (MHb) neurons was calculated from experiments that simultaneously monitored agonist-induced membrane currents and intracellular [Ca²⁺], measured with patch-clamp and indo-1 fluorescence, respectively. In physiological concentrations of extracellular Ca²⁺ (2 mM) at –50 mV, the percentage of current carried by Ca²⁺ was determined to be roughly 3–4%, which is in close agreement with measurements from other heteromeric nicotinic receptors expressed in peripheral tissue. Among factors that may have affected this measurement, such as Ca²⁺ influx through voltage-gated Ca²⁺ channels, the concentration of intracellular Ca²⁺ buffer, and Ca²⁺ sequestration and release from intracellular stores, only Ca²⁺ uptake by mitochondria was shown to confound the analysis. Furthermore, we find that because of the high density of nAChRs on MHb cells, low concentrations of ACh (10 µM) and its hydrolysis product, choline (1 mM), can significantly elevate intracellular Ca²⁺. Moreover, during persistent activation of nAChRs, the level of intracellular Ca²⁺ is proportional to its extracellular concentration in the physiological range. Together, these findings support the suggestion that nAChRs may be capable of sensing low concentrations of diffusely released neurotransmitter and, in addition, transfer information about ongoing local synaptic activity by changes in extracellular Ca²⁺.

	INTRODUCTION

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In addition to direct neuronal excitation by membrane depolarization, nicotinic acetylcholine receptors (nAChRs) flux Ca²⁺ and thereby contribute to intracellular signaling with a variety of functional consequences (Burnashev 1998; Rathouz et al. 1996). Included in these receptor roles are the Ca²⁺-dependent activation of K⁺ channels, resulting in hyperpolarization (Housley and Ashmore 1991; Tokimasa and North 1984; Wong and Gallagher 1991), the augmentation of intracellular Ca²⁺ by Ca²⁺-induced Ca²⁺ release (CICR) from cytoplasmic stores (Brain et al. 2001; Dajas-Bailador et al. 2002; Sharma and Vijayaraghavan 2001; Tsuneki et al. 2000), and the facilitation of transmitter release by presynaptically localized nAChRs (Gray et al. 1996).

The functional contribution of a diverse group of subunits produces a large variability in nicotinic receptor-mediated Ca²⁺ signaling (Colquhoun and Patrick 1997; Le Novere et al. 2002; McGehee and Role 1995; Role 1992). The relative Ca²⁺/monovalent ion permeabilities of nAChRs range from about 1 for heteromeric subtypes (Fieber and Adams 1991; Sands and Barish 1991; Vernino et al. 1992) to 20 for the highly Ca²⁺ permeable 7 receptor (Bertrand et al. 1993; Castro and Albuquerque 1995; Sands et al. 1993; Seguela et al. 1993). However, these Goldman–Hodgkin–Katz (GHK) reversal potential estimates of Ca²⁺ movement through nAChRs (Adams et al. 1980) do not always accurately predict the relative flux of Ca²⁺ under physiological conditions (Neher 1995; Rathouz et al. 1996; Vernino et al. 1994). Alternate quantitative measurements of fractional Ca²⁺ permeability using Ca²⁺–charge ratios obtained by simultaneously monitoring membrane currents and Ca²⁺ induced changes in indicator fluorescence have suggested that Ca²⁺ contributes 2.5–4.7% current flux through heteromeric receptors (Ragozzino et al. 1998; Rogers and Dani 1995; Vernino et al. 1994; Zhou and Neher 1993), whereas, in line with the larger Ca²⁺-mediated shifts in reversal potentials, Ca²⁺ contributes a significantly higher fraction of current (6.7–20%) through homomeric 7-type receptors (Delbono et al. 1997; Fucile et al. 2000, 2005).

Neurons in the medial habenula (MHb) possess a large population of Ca²⁺ permeable nAChRs (McCormick and Prince 1987; Mulle et al. 1992a), although the Ca²⁺ flux through these channels has not yet been quantified. Despite the presence of a wide variety of nicotinic receptor subunits within the MHb nucleus (Colquhoun and Patrick 1997), the majority of nAChRs have been pharmacologically defined as containing 3/4 subunits (Mulle and Changeux 1990; Quick et al. 1999) and thus appear to belong to the heteromeric set of receptors with moderate Ca²⁺ permeability. However, the presence of 5 (and other) subunits in these cells (Le Novere et al. 1996; Sheffield et al. 2000) may allow for a higher Ca²⁺ flux (Gerzanich et al. 1998). Thus it is important to quantify Ca²⁺ flux through native nAChRs on MHb neurons to test whether they conform, in terms of their Ca²⁺ permeability, to moderately or highly permeable receptors. In addition, aside from the studies on hippocampal neurons (Castro and Albuquerque 1995; Fayuk and Yakel 2005), all the above quantitative assessments of Ca²⁺ flux have been performed on nAChRs from peripherally derived tissue or from heterologously expressed channels. Based on these data we discuss the implications for subunit composition and possible physiological relevance of Ca²⁺ flux through this population of receptors. In particular, we reevaluate the importance of Ca²⁺ flux through nicotinic receptors in the MHb, which has so far been discounted because these nAChRs do not participate in conventional synaptic transmission (Edwards et al. 1992; Robertson et al. 1999), by exploring the idea that these receptors may function as sensors of ambient ACh and/or extracellular Ca²⁺.

	METHODS

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Acute isolation of habenula neurons

The medial habenula contains a morphologically homogeneous group of densely packed neurons that can be easily identified based on their distinct morphology (Mulle and Changeux 1990). In this study, neurons were isolated from rat medial habenula nuclei using methods described previously (Quick et al. 1999). In accordance with national guidelines, a single rat (10–20 days old) was anesthetized with halothane and decapitated. The habenula region was dissected in ice-cold PIPES buffer (containing, in mM: NaCl, 120; KCl, 5; MgCl₂, 5; PIPES, 25; D-glucose, 25; phenol red, trace; pH adjusted to about 7.2 with NaOH), and placed into 10 ml prewarmed PIPES buffer containing 90 U papain (Worthington Biochemical, Freehold, NJ), 10 mg bovine serum albumin (BSA), and 2 mg cysteine. Incubation was under 37°C for 25–30 min during which tissue was constantly agitated on a rocking shaker. The tissue was washed three times with PIPES buffer and once with a low-glucose Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) containing about 1 mg ml^–1 each of BSA and trypsin inhibitor. Neurons were dissociated from tissue in DMEM containing 1–2% fetal bovine serum with gentle trituration and plated onto poly-D-lysine (Collaborative Biochemical Products, Bedford, MA) coated (2 µg ml^–1) glass coverslips covering a central hole (diameter 15 mm) drilled through the base of 35-mm petri dishes. These petri dishes served as recording chambers. The cells were incubated at 37°C under 5% CO₂ for 8 h until use for recording. Unless otherwise stated, all chemicals were obtained from Sigma–Aldrich (St. Louis, MO).

Patch-clamp recording in acute isolated neurons

Petri dishes containing cells were mounted to the stage of an inverted microscope equipped for UV epifluorescence (Nikon Diaphot 200, Nikon, Tokyo, Japan) and continuously superfused with a standard extracellular medium containing (in mM): NaCl, 150; HEPES, 10; D-glucose, 10; CaCl₂, 2 (pH 7.4). The extracellular "pure" Ca²⁺ solution used for calibration contained (in mM): HEPES, 10; D-glucose, 10; CaCl₂, 75 [pH was adjusted to 7.4 with Ca(OH)₂ and osmolarity was adjusted to 300 mOsmol with sucrose]. Medial habenula (MHb) neurons (Quick et al. 1999) were visualized with a x40 (NA = 1.3) oil-immersion lens. Whole cell patch-clamp recordings were made using fire-polished glass pipettes (#7052; Garner Glass, Clairmont, CA), filled with a filtered internal solution containing (in mM): N-methyl-D-glucamine (NMDG), 140; tetraethylammonium chloride, 5; Mg-ATP, 1; HEPES, 20; EGTA, 0.1–0.4; and indo-1 pentapotassium, 0.05 (pH 7.2). The intracellular solution was designed to allow a one-way influx of ions (see Vernino et al. 1994). Pipettes had open-tip resistances of 2–5 M and series resistance (R_S) was compensated 80%. Recordings with R_S >20 M were rejected. Currents were recorded with Axopatch 1-D amplifier (Axon Instruments, Foster City, CA), low-pass filtered, and digitally sampled at 250 Hz. The relatively low sampling rate was necessary to allow interleaved sampling of the voltage output from the two photomultiplier tubes (see following text). The holding potential for all experiments was –50 mV.

Drug application

Control and drug-containing solutions were gravity fed into a linear array of glass barrels (ID = 0.38 mm; Garner Glass) positioned close (<100 µm) to the neuron. The barrels were attached to a piezo-electric bimorph (Piezo Systems, Cambridge, MA), connected to a variable 0- to 120-V DC-power source under computer control, and could be rapidly repositioned. Complete exchange of solutions occurred in about 50 ms (Lester and Dani 1995) and, estimated from the decay rate of currents after the stop of agonist application, drugs around the recorded neuron were completely washed away within 1 s.

Intracellular calcium measurements

Indo-1 (Molecular Probes, Eugene, OR) was excited at 365 nm using 1% light generated by a 100-W mercury arc lamp. Emitted light >380 nm was initially captured through a dichroic mirror (Chroma Technology, Brattleboro, VT) and then split using a second 440-nm dichroic mirror to two separate photomultiplier tubes (PMT; D-104 photometer, Photon Technology International, Lawrenceville, NJ) with input filters at 405 and 485 nm. Emissions were collected from the cell soma only by means of an adjustable aperture. The PMT signals were integrated over 5 ms and the output voltages of the integrators were sampled at 250 Hz, interleaved with the whole cell current, using the Digidata-1200 (Axon Instruments). Axobasic software (Axon Instruments) was used for on-line display of the raw voltage outputs in addition to the ratio (405 nm/490 nm) of the two signals. Sensitivities of the PMTs and the output voltages were adjusted to give a baseline ratio of about 0.5. For monitoring indo-1 loading of cells and resting intracellular Ca²⁺, cells were generally excited for 100-ms periods every 2 s using a Uniblitz shutter (Vincent Associates, Rochester, NY) under computer control. Background fluorescence was offset in cell-attached mode. For agonist-evoked responses, the shutter was timed to open 500–1,000 ms before the application of drug. The typical exposure length of 4 s induced negligible bleaching of the dye, as judged from the raw PMT output signals. In addition, applications of agonist were made at sufficient intervals (3 min) for the fluorescence ratio to return to resting levels. In general <1 min was required for the transient rise in intracellular [Ca²⁺] to recover (see Fig. 1D) and cells that required more than 3 min were discarded.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Simultaneous measurement of nicotinic acetylcholine receptor (nAChR)–mediated currents and intracellular Ca2+ ([Ca2+]i). A: example of the increase in [Ca2+]i (top trace) and inward current in response to the application of 30 µM nicotine for 1 s (bottom trace). Dashed lines indicate the start and end of the drug exposure. B: plot of the change in intracellular [Ca2+] ([Ca2+]i, calculated by subtraction of the resting intracellular [Ca2+] from the instantaneous [Ca2+]) with respect to the inward charge movement (calculated by integration of the current, as indicated by shaded area in A; see text). Arrows in A and B indicate the peak of the current. C: plot of the [Ca2+]i vs. the change in the raw fluorescence ratio (ratio; 405 nm/490 nm). D: example of rise and decay of [Ca2+]i during and after a nicotine application (2 s; 100 µM). E: membrane currents (bottom traces) and [Ca2+]i (top traces) in response to nicotine (30 µM; 1 s) in the presence (left) and absence (right) of extracellular Ca2+ ([Ca2+]o). [Ca2+]i is expressed as the raw fluorescence ratio (405 nm/490 nm).

Estimates of intracellular [Ca²⁺] were obtained using the R_min/R_max method (Grynkiewicz et al. 1985)

(1)

where R is the ratio of fluorescence at 405 nm/490 nm and R_min and R_max are the minimum and maximum values of the ratio obtained by perfusing cells with EGTA (1 mM) and pure (75 mM) Ca²⁺, respectively, in the presence of ionomycin (5 µM). K_d is the dissociation constant for indo-1, taken as 250 nM (Grynkiewicz et al. 1985) and S_f2 and S_b2 are the upper and lower limits of the raw fluorescence signal measured at 490 nm.

The Ca²⁺-flux ratio (F/q) for MHb nAChRs was calculated from a point-to-point comparison of F and q, where the Ca²⁺ (F) was the instantaneous increase in intracellular [Ca²⁺] from its resting level at time t, and the charge flux (q) was obtained by integrating the area under the nicotine-induced current up to t, where t can be part of or the entire duration of the agonist application (Fig. 1, A and B). In four cells, in which the [Ca²⁺] was determined from Eq. 1, the mean intracellular [Ca²⁺] after application of nicotine (30 µM) peaked at 162 ± 49 nM from a resting level of 78 ± 32 nM (Mulle et al. 1992a) and the F/q ratio was 211 ± 33 nM/nC. Accurate determination of the F/q ratio is dependent on exact correspondence between the change in intracellular [Ca²⁺] and the current flux through the open channels. The presence of nAChRs on dendrites would lead to an underestimation of the F/q ratio because such channels would give rise to additional ion influx but contribute little to the change in [Ca²⁺] measured somatically. In acutely isolated MHb cells, however, there will be close agreement between the measured current and the [Ca²⁺] change because these are small cells (soma diameter 15 µm), largely devoid of dendritic processes.

Although in some cells as noted above, we converted the fluorescence ratio into the concentration of intracellular Ca²⁺, this calculation was not performed in every case because most of the experiments in this study were not dependent on absolute [Ca²⁺] but required only a comparison of the relative fluorescence–charge (F/q) ratios under different conditions (where F is the fluorescence ratio at 405 nm relative to 490 nm; see Vernino et al. 1994). The only provision in this case would be that the change in intracellular [Ca²⁺] is linearly related to the fluorescence ratio (Fig. 1C). One benefit of the uncalibrated approach is that it allows for small adjustments to the sensitivity of the PMTs to compensate for differential dye loading between cells, without the need for calibration in every cell.

Statistical analysis

Unpaired Student's t-tests were used to compare the percentage of current carried by Ca²⁺ ions under different recording conditions. Paired Student's t-tests were used in all other comparisons. Significance was P < 0.05. Ca²⁺–charge slopes were fit by linear regression using a least-squares method. Data are presented as means ± SE.

Receptor nomenclature

Subtypes of nAChRs are referred to by their putative subunit composition with an asterisk to represent likely inclusion of additional subunits (Lukas et al. 1999).

	RESULTS

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Ca²⁺ flux through MHb nicotinic channels

Activation of nicotinic receptors in MHb neurons was previously shown to significantly elevate intracellular [Ca²⁺] (Mulle et al. 1992a). The goal of the present study was to quantify the Ca²⁺ flux for comparison with other heterologously and natively expressed nAChRs. Toward this end, it is necessary to first show that the measured increase in intracellular Ca²⁺ arises as a direct result of channel activation. If this condition is satisfied then there should be a linear relationship between the charge influx and the change in intracellular [Ca²⁺]. In MHb cells, the Ca²⁺–charge (F/q) ratio, obtained from simultaneous current and [Ca²⁺] measurements (Fig. 1A), is linear over the entire duration of the response (Fig. 1B). Deviations from linearity could indicate, e.g., Ca²⁺-dependent Ca²⁺ release (Brain et al. 2001; Dajas-Bailador et al. 2002; Sharma and Vijayaraghavan 2001; Tsuneki et al. 2000) or saturation of the Ca²⁺ indicator and/or other buffers. During experiments, intracellular [Ca²⁺] must return to its resting level after an agonist exposure to ensure a similar saturation of the Ca²⁺ indicator before the next exposure. To study the time course of the decay of intracellular [Ca²⁺] after agonist applications, a high concentration of agonist (100 µM nicotine; 2 s) was applied to the neurons (Fig. 1D). In these experiments, intracellular [Ca²⁺] increased from a resting level of 83 ± 11 nM to a peak of 870 ± 189 nM and the decay process of intracellular [Ca²⁺] was best fitted to a single-exponential component with a time constant of 7.7 ± 1.3 s (n = 5), indicating that the Ca²⁺ changes measured during the F/q experiments were at the low end of the concentration range that can be effectively handled by the cells.

The increase in [Ca²⁺] should be completely dependent on the presence of extracellular Ca²⁺ if it is the result of entry through plasma membrane nAChRs. Figure 1E shows a comparison between nicotinic currents recorded in the presence of 2 mM extracellular Ca²⁺ and a nominally Ca²⁺ free solution (n = 6). Removal of extracellular Ca²⁺ eliminated the increase in fluorescence, implying that the observed Ca²⁺ increases are dependent on extracellular Ca²⁺ influx. In addition, the current amplitude in the presence of extracellular Ca²⁺ was significantly greater than that in Ca²⁺-free external solution (1,337 ± 183 and 1,180 ± 164 pA, respectively; n = 6; P < 0.05), consistent with the reports that extracellular Ca²⁺ can potentiate current through nAChRs (Mulle et al. 1992b; Vernino et al. 1992). Taken together with the linear Ca²⁺–charge relationship, these data imply that the increase in intracellular Ca²⁺ is solely attributable to the influx of Ca²⁺ through open nAChR channels. However, this result alone does not definitively demonstrate that other cellular processes, such as buffering and store-released Ca²⁺, do not contribute to the changes in intracellular Ca²⁺. These factors, which could complicate interpretation of F/q data, are considered below.

Fraction of current carried by Ca²⁺

Several methods were previously used for determination of the Ca²⁺ permeability of nAChRs, including the classical GHK approach based on shifts in reversal potentials (Rathouz et al. 1996). With this method, however, it is difficult to predict the fractional flux of Ca²⁺ through channels under physiological conditions and alternate approaches, based on direct measurements of intracellular [Ca²⁺], were developed (Vernino et al. 1994; Zhou and Neher 1993). Normalization of the F/q ratio in physiological extracellular solution with the F/q ratio obtained under conditions in which all the charge is carried by Ca²⁺ is performed using the equation

(2)

where (F/q)2Ca²⁺ and (F/q)75Ca²⁺ are the Ca²⁺–charge ratios measured in the presence of 2 mM Ca²⁺ and pure Ca²⁺ (75 mM), respectively. Because each cell acts as its own control, errors arising from cell-to-cell differences in the magnitude of Ca²⁺ transients as a result of differential cell size and/or buffering will be effectively eliminated (Vernino et al. 1994). In addition, this method is particularly useful because it will allow comparison of the fractional Ca²⁺ flux (FCa²⁺) measured in different sets of studies on heterologously expressed and native receptors.

Figure 2 shows examples of the experimental approach used in the present study. Cells were initially exposed to nicotine (30 µM) in the presence of 2 mM extracellular Ca²⁺ and the resulting current and change in intracellular [Ca²⁺] were recorded (Fig. 2A, left). After several applications (generally three to four) in 2 mM Ca²⁺, for the purpose of data averaging, nicotine (30 µM) was applied in pure (75 mM) extracellular Ca²⁺, after a transient (500-ms) period in Na⁺-free (NMDG⁺-substituted) solution to ensure that Ca²⁺ would be the only permeative specie during the nicotine-induced inward current (Fig. 2A, right). Currents in pure Ca²⁺ are much smaller than those in physiological solution because, despite being both permeable to and potentiated by Ca²⁺, channel conductance is reduced in the presence of this divalent (Amador and Dani 1995; Mulle et al. 1992b). Conversely, the elevation in intracellular [Ca²⁺] is more rapid and much larger, as expected, for a higher Ca²⁺ influx. As a consequence of recording under these two conditions the calculated F/q ratio for pure Ca²⁺ is much greater than that in physiological Ca²⁺ (Fig. 2B). The mean percentage inward current carried by Ca²⁺ ions, by application of Eq. 2 to the relative F/q ratios, was 4.2 ± 0.4% (n = 10).

View larger version (16K): [in this window] [in a new window]   FIG. 2. Charge-flux comparisons in different extracellular [Ca2+]. A: simultaneous measurement of intracellular [Ca2+] and membrane current in 2 mM (left) and pure 75 mM (right) [Ca2+]o-containing solutions. B: plots of the [Ca2+]i (expressed as a change in raw fluorescence ratio) with respect to the inward charge movement (as indicated by the shaded areas in A, only part of area under the membrane currents was integrated) in physiological extracellular (circles) and pure [Ca2+]o (squares).

Potential factors that may confound estimates of the fractional current carried by Ca²⁺ include cytoplasmic Ca²⁺ buffering in addition to the sequestration and release of Ca²⁺ from intracellular stores. Although the F/q ratio in physiological solution is normalized to the Ca²⁺–charge in pure Ca²⁺, substantial errors in the calculation of FCa²⁺ could arise if there were a differential effect of buffering in the two recording conditions. In the case of Ca²⁺ buffering, it is conceivable that added EGTA (0.2 mM) could dampen the Ca²⁺ transient to a greater extent in physiological solution (2 mM Ca²⁺) compared with pure Ca²⁺ (75 mM). This would lead to an underestimation of the fractional Ca²⁺ flux. To address this issue the concentration of intracellular EGTA was varied from 0.1 to 1 mM. At 1 mM EGTA, only small fluorescent changes in the presence of nicotine were observed (data not shown), implying that there was sufficient EGTA to fully compete with the fluorescent dye. However, varying the concentration of EGTA from 0.1 to 0.4 mM did not affect the estimation of FCa²⁺, thereby demonstrating that differential Ca²⁺ buffering was not a confounding issue (Fig. 3A). Although space-clamp problems are unlikely in these small, round, acutely isolated cells, another potential source of Ca²⁺ is by voltage-gated Ca²⁺ channels that are activated by any unclamped depolarization resulting from nAChR activation. This concern was addressed by adding 200 µM Cd²⁺ to the external solution. This concentration of Cd²⁺ effectively blocks both the inward current and intracellular Ca²⁺ transient evoked by depolarizing voltage steps (Guo and Lester 2007). In the presence of Cd²⁺, the percentage Ca²⁺ influx (5.0 ± 0.4%; n = 5) was not significantly different from control (4.2 ± 0.4%; n = 10; P > 0.05; Fig. 3A).

View larger version (19K): [in this window] [in a new window]   FIG. 3. Effects of different factors on the measured fractional Ca2+ flux. A: histogram showing the measured fraction of current carried by Ca2+ under different intra- and extracellular conditions. Ruthenium red (RR, 20 µM) and cyclopiazonic acid (CPA, 6 µM) were added to the intracellular solution and 200 µM Cd2+ was added to the extracellular solution. B, left: changes in [Ca2+]i induced by 2 applications of caffeine (10 mM) under control conditions (top traces) and before and after a 5-min bath application of 1 µM thapsigargin (bottom traces). [Ca2+]i is expressed as the raw fluorescence ratio (405 nm/490 nm). Right: histograms of the normalized Ca2+ responses (2nd/1st) in control and thapsigargin-treated cells. C and D: fractional Ca2+ influx measured in the same cell before and after bath application of thapsigargin or FCCP. Number of cells in each condition is shown in parentheses. *P < 0.05.

Cytosolic Ca²⁺ can also be rapidly (<1 s) sequestered by mitochondria (Pivovarova et al. 2002) on a time course similar to our applications of nicotine and could lead to an underestimation of the F/q ratios, particularly in high [Ca²⁺]. Therefore we included ruthenium red (20 µM) in the pipette solution to block the uniporter responsible for Ca²⁺ uptake into mitochondria (Ying et al. 1991). Ruthenium red (RR) significantly reduced our estimation of the fractional current carried by Ca²⁺ from a control level of 4.2 ± 0.4 (n = 10) to 3.1 ± 0.4 (n = 4; P < 0.05) (Fig. 3A). To confirm these results, bath application of the well-known mitochondrial inhibitor carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was used so that the FCa²⁺ measurements could be compared in the same cell. FCCP, in a similar manner to RR, produced a decrease in the measured fractional Ca²⁺ influx from 4.6 ± 0.3 to 3.6 ± 0.5 (n = 6; P < 0.05) (Fig. 3D). These data seem most readily explained if the F/q ratio was increased more by RR and FCCP in pure Ca²⁺ solutions compared with the physiological solution, a suggestion substantiated by the low-affinity high-capacity nature of mitochondria for Ca²⁺, which would be more effective after a rapid and considerable rise in intracellular Ca²⁺ (Verkhratsky and Peterson 1998). In agreement with this explanation, the F/q ratios (in arbitrary units) in 2 mM Ca²⁺ were comparable in the presence and absence of FCCP (0.77 ± 0.12 and 0.73 ± 0.12; n = 6; P > 0.05), whereas there was a trend, albeit insignificant, toward an increased F/q ratio after the treatment of FCCP in the pure Ca²⁺ external solution. The ratios were 17 ± 3 and 21 ± 4 (n = 6; P > 0.05) before and after FCCP, respectively.

Elevation of intracellular Ca²⁺ by endogenous agonists

Synaptic activation of nAChRs by endogenous acetylcholine (ACh) in the MHb has not been demonstrated (Edwards et al. 1992), leaving the role of these ion channels unclear. Furthermore, it has been argued that the major, and probably only, physiological source of Ca²⁺ influx in MHb cells is by purinergic receptors (Robertson et al. 1999). An explanation for the high density of Ca²⁺-permeable nAChRs on these neurons (Mulle et al. 1992a) is therefore warranted. One possibility is that these receptors are stimulated by diffusely released ACh, or its hydrolysis product choline (Alkondon et al. 1997; Zoli et al. 1999). If this is the case, then receptors may be exposed only to low concentrations of agonist. The EC₅₀ for ACh on MHb cells is 80 µM (Mulle and Changeux 1990), so a concentration of 10 µM will activate about 5% of the total receptor population, but because of the high channel density (Lester and Dani 1994), the absolute number of receptors activated could be substantial. We tested whether a low concentration of ACh was effective in increasing intracellular Ca²⁺ in MHb cells. Figure 4A shows that a 10-s application of 10 µM ACh can significantly raise the cytosolic [Ca²⁺]. The mean peak [Ca²⁺] was increased to 157 ± 14 nM from a resting level of 87 ± 4 nM (n = 3; P < 0.05). ACh, however, may not be a viable diffuse signal because it is rapidly hydrolyzed in the MHb (Quick et al. 1999). In this respect, its breakdown product, choline, may more readily function in a paracrine manner. Choline acts as a weak partial agonist at the 34* subtype of nAChR (Alkondon et al. 1997; Papke et al. 1996) and, at a concentration of 1 mM, could both induce a nicotinic current (Parker et al. 2003) and elevate intracellular Ca²⁺ to a similar level as observed with ACh (Fig. 4B). In this case the intracellular [Ca²⁺] increased from a resting level of 82 ± 5 to 158 ± 10 nM (n = 4; P < 0.05). Thus moderate increases in intracellular Ca²⁺ can be induced by nicotinic agonists in a manner consistent with a diffuse mode of receptor activation.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Changes in intracellular [Ca2+] induced by physiological agonists. Examples of changes in intracellular [Ca2+] and membrane currents induced by 10-s applications of 10 µM acetylcholine (ACh, A) and 1 mM choline (B). [Ca2+]o = 2 mM. Membrane currents induced by choline (10 mM; 1 s) in medial habenula (MHb) neurons in the absence (left traces) and presence (right traces) of dihydro-beta-erythroidine (DHE, 10 µM; C) and methyllycaconitine (MLA, 5 nM; D). Histograms of the mean data are shown on the right and the number of cells in each condition is shown in parentheses. *P < 0.05.

nAChRs report changes in extracellular Ca²⁺

In the narrow extracellular space inside the brain, extracellular [Ca²⁺] fluctuates dramatically during neuronal activity (Heinemann et al. 1977; Stabel et al. 1990). Because low concentrations of agonists can induce prolonged activation of slowly desensitizing nAChRs in the MHb (Lester and Dani 1995), one function of these receptors could involve detection of changes in extracellular [Ca²⁺]. The mechanistic feasibility is illustrated in Fig. 5. The nAChRs were first activated by 10 µM ACh in 0.5 mM extracellular Ca²⁺ for 5 s, after which the extracellular Ca²⁺ was changed to 4 mM in the continued presence of ACh for an additional 5 s (Fig. 5A). After application of ACh, intracellular [Ca²⁺] increased from a resting level of 110 ± 22.9 nM to a plateau of 123 ± 23 nM (P < 0.05) in 0.5 mM extracellular Ca²⁺ and was further elevated to 153 ± 24 nM (P < 0.05) in 4.0 mM extracellular Ca²⁺ (n = 5; Fig. 5B). In these experiments, there was no significant change in the amplitude of nAChR-mediated currents when extracellular [Ca²⁺] was increased (see Fig. 5A). This lack of potentiation by extracellular Ca²⁺ was observed in previous studies on MHb cells and was attributed to intracellular dialysis (Hicks et al. 2000). Despite the expectation that there should be a increase, albeit small, in current amplitude based solely on the additional charge in 4 mM [Ca²⁺], the linear F/q relationship was maintained throughout the application, although the slope was predictably increased when [Ca²⁺] was switched from 0.5 to 4.0 mM (Fig. 5D). In cells (n = 3) that failed to respond to ACh and therefore serve as controls, changing extracellular [Ca²⁺] from 0.5 to 4.0 mM did not affect the resting level of intracellular Ca²⁺ (Fig. 5C).

View larger version (26K): [in this window] [in a new window]   FIG. 5. Intracellular [Ca2+] follows extracellular [Ca2+] during persistent activation by ACh. A: changes in intracellular [Ca2+] and inward current during exposure to different extracellular [Ca2+]. Cells were initially exposed to 10 µM ACh in a low-[Ca2+]o–containing external solution (0.5 mM) and then to a high-[Ca2+]o–containing external solution (4 mM) in the same concentration of ACh. Arrows indicate the timing of the change from low to high external [Ca2+]. B: mean intracellular [Ca2+] at rest and in low and high [Ca2+]o during exposure to ACh (n = 5). *P < 0.05. C: example of membrane current (bottom trace) and intracellular Ca2+ (top trace) from a cell that failed to respond to ACh. [Ca2+]i is expressed as the raw fluorescence ratio (405 nm/490 nm). D: plot of [Ca2+]i vs. charge influx for the same cell as shown in A. Data obtained in 0.5 mM (solid circles) and 4 mM (open circles) external Ca2+ solutions were fit separately with 2 linear regressions [correlation coefficient (r) = 0.68 and 0.94, respectively]. The relative slopes of the lines were 0.013 and 0.050 nM/pC.

	DISCUSSION

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nAChR subtype-dependent calcium permeability

The relative calcium permeabilities of neuronal nAChRs fall into two major categories: moderate, for heteromeric receptors; and high, for -bungarotoxin (BTX)-sensitive homomeric subunit containing only receptors (Burnashev 1998; Rathouz et al. 1996). Based on mRNA expression (Dineley-Miller and Patrick 1992; Duvoisin et al. 1989; Wada et al. 1989; Winzer-Serhan and Leslie 1997), for radiolabeled ligand-binding sensitivities (Marks et al. 1998; Perry and Kellar 1995; Zoli et al. 1998) and functional pharmacology (Mulle and Changeux 1990; Quick et al. 1999) the subunit composition of MHb receptors has been determined to be largely 34 like. With this information alone, we would predict a moderate calcium permeability for these receptors. To aid our analysis, there have been a number of studies on both heterologously expressed and native nicotinic channels, permitting a detailed comparison of Ca²⁺ permeabilities.

The most conservative estimate of Ca²⁺ flux in the present study comes from experiments in which Ca²⁺ uptake into mitochondria was blocked by RR and FCCP. We hypothesize that rapid Ca²⁺ buffering by mitochondria decreased the F/q ratio in pure extracellular Ca²⁺ and therefore led to an overestimation of the fraction of current carried by Ca²⁺ (FCa²⁺ = 4.2%). Block of the mitochondrial Ca²⁺ uptake reduced the estimate of FCa²⁺ to 3.1–3.6%. This value is within the range of fractional Ca²⁺ fluxes for various chick receptors expressed in BOSC 23 cells (Ragozzino et al. 1998). In their study, however, Ca²⁺ contributed 4.6% inward current through 34 receptors—the highest for any of the heteromeric nAChRs tested. Provided that rat and chick receptors are similar, and that their estimate of FCa²⁺ is not confounded by Ca²⁺ sequestration, then these data imply that MHb nAChRs may not simply be composed of 3 and 4 subunits. In particular, it is noted that 4- and 2-subunit–containing receptors have lower (about 3%) fractional Ca²⁺ flux contributions (Ragozzino et al. 1998) and both of these subunits could potentially contribute to the heterogeneity of nAChRs in the MHb (Connolly et al. 1995; Quick et al. 1999; Sheffield et al. 2000).

In cells derived from the peripheral nervous system that likely express receptors containing at least 3 and 4 subunits (Sargent 1993), the FCa²⁺ values are 4–10% [superior cervical ganglion (SCG) cells; Trouslard et al. 1993], 2.5% (chromaffin cells; Zhou and Neher 1993), 4.1% (chromaffin cells; Vernino et al. 1994), 4.8% (Rogers et al. 1997), and 2.2% (Fucile et al. 2005). It seems likely that some of the differences probably resulted from disparate experimental conditions; in the study producing the lowest estimates of FCa²⁺, for example, the F/q ratio in physiological solution was normalized using calcium currents, not nAChRs in pure Ca²⁺ (Zhou and Neher 1993). In addition, the higher extracellular [Ca²⁺] (2.5 mM) used in some of the previous studies may have accounted for their high FCa²⁺. To facilitate a more accurate comparison, previously published FCa²+[r] values for 34* receptors from peripheral tissue were normalized to 2 mM external [Ca²⁺] (Table 1). The FCa²⁺ for nAChRs in MHb neurons (3.1–4.2%) falls in the midrange of reported values. In conclusion, our quantitative FCa²⁺ estimate is in close agreement with these studies, consistent with the idea that similar heteromeric nAChRs are expressed in both the MHb and autonomic tissues.

View this table: [in this window] [in a new window]   TABLE 1. Comparison of Ca2+ for putative 34* receptors in previous studies

Can nAChRs detect ambient ACh and activity-dependent fluctuations in extracellular [Ca²⁺]?

Despite an obvious relevance in tobacco addiction (Mansvelder and McGehee 2002), a clear view of the physiological relevance of nAChRs in the brain is lacking. This problem is exemplified in the MHb–interpeduncular (IPN) tract (Brown 2000). Not only are nAChRs found somatically in the MHb, they are also present in the fasciculus retroflexus (FR), presumably MHb axons projecting to their termination in the IPN (Marks et al. 1998; Perry and Kellar 1995). Moreover, the somatic nAChRs on IPN cells are not synaptically activated by electrical stimulation of the FR, which should contain the appropriate cholinergic input (Brown et al. 1983). Likewise, the high density of nAChRs on MHb cell bodies (Lester and Dani 1994) do not appear to sense any rapidly released ACh (Edwards et al. 1992). Thus the significance of such receptors remains unclear. One possible explanation is that these receptors are not stimulated by synchronously released transmitter, but are activated more slowly by ambient ACh in a diffuse manner (Zoli et al. 1999). In this respect, we find that low concentrations of this transmitter (and higher concentrations of its metabolite, choline) can significantly elevate intracellular Ca²⁺ and suggest that one role for MHb nAChRs may be to sense fluctuations in ambient [ACh] by changes in intracellular Ca²⁺, in addition to, or as an alternative to a change in membrane voltage.

Within the confined extracellular space, [Ca²⁺] fluctuates dramatically during synaptic activity (Heinemann et al. 1977; Stabel et al. 1990) and may encode important information that can be detected by a variety of mechanisms. In synaptic clefts and surrounding areas, a decrease in [Ca²⁺] was shown to directly affect synaptic transmission (Borst and Sakmann 1999; Stanley 2000). Furthermore, extracellular Ca²⁺ sensing receptors (Brown et al. 1993) and metabotropic glutamate receptors (Kubo et al. 1998) are also capable of detecting changes in extracellular [Ca²⁺] in the CNS. With respect to nAChRs, changes in extracellular [Ca²⁺] can also be sensed by an allosteric change in channel open probability (Amador and Dani 1995; Mulle et al. 1992b). Here we suggest that, in the continuous presence of a low concentration of ACh, variations in extracellular [Ca²⁺] can induce corresponding changes in intracellular [Ca²⁺]. In theory this would allow direct coupling between changes in local synaptic neuronal activity and various Ca²⁺-dependent intracellular processes. An obvious effector mechanism is the Ca²⁺-dependent K⁺ channel, which can couple nAChRs to a physiologically important membrane hyperpolarization (Glowatzki and Fuchs 2000; Housley and Ashmore 1991; Tokimasa and North 1984; Wong and Gallagher 1991). In terms of MHb function, persistently open nAChR channels could translate alterations in extracellular [Ca²⁺] into a change in the firing pattern of these spontaneously active neurons (McCormick and Prince 1987), by the highly expressed slo K⁺ channels (Knaus et al. 1996), potentially influencing rhythmic behaviors such as REM sleep (Haun et al. 1992). Conversely, under conditions such as chronic nicotine, strong Ca²⁺ influx through slowly desensitizing nAChRs, may explain the selective degeneration of MHb cells and their axons (Carlson et al. 2000).

	GRANTS

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This work was supported by National Institute of Neurological Disorders and Stroke/Public Health Service Grant NS-31669.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R.A.J. Lester, Department of Neurobiology, SHEL1006, University of Alabama at Birmingham, 1825 University Boulevard, Birmingham AL 35294-2182 (E-mail: rlester{at}nrc.uab.edu)

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