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Norepinephrine acts via α2 adrenergic receptors to suppress N-type calcium channels in dissociated rat median preoptic nucleus neurons
Neuropharmacology 41 (2001) 472–479 www.elsevier.com/locate/neuropharm
Norepinephrine acts via α2 adrenergic receptors to suppress N-type calcium channels in dissociated rat median preoptic nucleus neurons M. Kolaj, L.P. Renaud
*
Neurology and Neurosciences, Loeb Health Research Institute, Ottawa Hospital —- Civic Site and University of Ottawa, 1053 Carling Avenue, Ottawa, Ont., Canada K1Y 4E9 Received 2 April 2001; received in revised form 25 April 2001; accepted 14 June 2001
Abstract The median preoptic (MnPO) nucleus, a key CNS site for hydromineral and cardiovascular homeostasis, receives a dense norepinephrine innervation from brainstem autonomic centers. Since norepinephrine is known to influence neuronal excitability by modulating calcium channel function, we applied whole cell patch clamp techniques to study calcium currents in 116 dissociated MnPO neurons, including 30 cells identified by a retrograde label as projecting to the hypothalamic paraventricular nucleus. Norepinephrine (3–50 µM) suppressed high-voltage-activated calcium currents (HVA ICa) in 80% of cells, selectively blockable by yohimbine and mimicked by UK14,304 and clonidine. The norepinephrine effect was relieved by strong prior depolarization, indicating a voltagedependent component. Intracellular GTP-γ-S blocked the effect. Blockade by extracellular NEM suggested involvement of pertussistoxin sensitive G-proteins. Based on pharmacological properties, these HVA ICas had the following composition: 40–45% N-type (blockable by ω-conotoxin GVIA); 20–25% L-type (blockable by nimodipine); 15–20% P/Q-type (blockable by ω-agatoxin IVA). Since 苲75% of the norepinephrine effect was blockable with ω-conotoxin GVIA, we conclude that postsynaptic α2 adrenoceptors preferentially suppress N-type calcium channels, revealing a novel mechanism whereby norepinephrine can modulate excitability in MnPO neurons. 2001 Elsevier Science Ltd. All rights reserved. Keywords: High voltage calcium currents; N type channels; Median preoptic nucleus; Norepinephrine; Dissociated cells; Patch clamp technique
1. Introduction Calcium is an important participant in the control of many neuronal functions, including regulation of their excitability, and voltage-dependent Ca2+ channels provide one of the many routes of calcium entry into neurons. Initially these channels were classified as low- and high-voltage-activated (LVA and HVA) currents on the basis of their differential activation and inactivation properties (Carbone and Swadulla, 1993). Multiple subtypes of HVA calcium currents (HVA ICa) can now be identified on the basis of their electrophysiological and pharmacological characteristics: nimodipine- or dihydropyridine-sensitive L-type currents, ω-conotoxin GVIA-
* Corresponding author. Tel.: +1-613-761-5070; fax: +1-613-7615360. E-mail address: [email protected] (L.P. Renaud).
sensitive N type currents, ω-agatoxin IVA-sensitive P/Q type currents, and a residual R-type that is insensitive to channel antagonists. Importantly, a wide variety of neurotransmitters and peptides, including norepinephrine, can be shown to modulate (usually inhibit) voltage-dependent Ca2+ channels (Brown and Birnbaumer, 1990; Anwyl, 1991; Hille, 1994). The characterization of calcium currents and their modulation are important for understanding the function of central neurons, including those comprising the median preoptic nucleus (MnPO), a parvocellular group of cells located at the midpoint of the lamina terminalis forming the anterior wall of the third cerebral ventricle. A variety of whole animal studies attest to the importance of the MnPO in achieving behavioral and endocrine responses to intracerebroventricular (icv) angiotensin, and in maintaining hydromineral homeostasis (Johnson et al., 1996; McKinley et al., 1996). Lesions of the MnPO severely disrupt salt and water balance,
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cardiovascular and behavioral responses to icv angiotensin (Mangiapane et al., 1983; Gardiner et al., 1985; Cunningham and Johnson, 1989; Xu and Hebert, 1995). There is reason to suspect that the brainstem innervation to this region is also critical to normal function. MnPO is the recipient of a prominent catecholaminergic (i.e. norepinephrine) input arising from the medulla (Saper and Levisohn, 1983; Kawano and Masuko, 1993). Neurotoxin-induced lesions of these catecholaminergic fibers markedly disrupt both hydromineral balance and angiotensin-induced drinking behavior, events that can be restored with norepinephrinergic transplants or infusions (McRae-Degueurce et al., 1986; Cunningham and Johnson 1989, 1991). Interestingly, similar deficits follow icv pretreatment with HVACa channel antagonists (Zhu and Herbert, 1997). However little is known of the neuronal mechanisms operative within this nucleus, including the intrinsic cellular properties and the neuropharmacology of its constitutive neurons. As an initial approach, we have used whole cell patch clamp technique in brain slices to begin a characterization of the intrinsic properties unique to MnPO neurons, and to evaluate their transmitter receptors. In a recent analysis (Bai and Renaud, 1998), we noted that 苲60% of neurons responded to bath applied norepinephrine with a postsynaptic α2-mediated membrane hyperpolarization or an α1-mediated membrane depolarization, both likely involving changes in potassium ionic conductances (Bai and Renaud, 1998). To extend this analysis, we sought to focus on MnPO neurons identified as projecting their axons to a known neuroendocrine and autonomic target of MnPO neurons i.e. the hypothalamic paraventricular nucleus or PVN (Silverman et al., 1981). Taking advantage of retrograde labeling from PVN and the resolution of dissociated cell preparations of MnPO, we have addressed the hypothesis that norepinephrine could also have a modulating influence on their calcium currents. We now report that L, N and P/Q type channels contribute to HVA ICa in MnPO neurons, and that norepinephrine selectively inhibits their N-type channels. A preliminary account of these results has been presented (Kolaj et al., 2000).
2. Methods 2.1. Preparation Adult male Long–Evans rats (60–120 g body wt) were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Animals were initially anesthetized with Somnotol (50 mg/kg), placed in a stereotaxic frame and a burr hole drilled on each side of midline to allow insertion of a micropipette. Animals received 0.2 µl of a suspension of a retrograde tracer (rhodamine latex microspheres; Molecular Probes Inc.) delivered by stereotaxic microinjection over 8–
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10 min into each hypothalamic paraventricular nucleus (coordinates from bregma: ⫺1.0, lateral 0.5, depth 6.5– 7.0). After closure of the skin with nylon sutures, animals were returned to individual cages. About two to seven days later, animals were sacrificed by decapitation, the brain quickly removed and placed in cooled (4°C) gassed (O2) PIPES-buffered solution (PBS, in mM: NaCl 120, KCl 5, glucose 25, CaCl2 1, MgCl2 1, PIPES 20 and NaOH for pH 7.1). The brain was blocked and sectioned at 250–300 µm with a vibratome, retaining the section that contained the MnPO and anterior commissure. The MnPO area was punched out and placed in an oxygenated bathing solution (PBS) containing pronase (0.1 mg/ml, Calbiochem) for 15 min. After transfer to a solution containing thermolysin (0.1 mg/ml, Sigma) for 15 min at 35°C, the tissue section was rinsed in PBS, then washed several times in a HEPES-buffered solution (HBS) containing (in mM) NaCl 150, KCl 5, glucose 10, CaCl2 2, MgCl2 1, Hepes 10 at pH 7.3–7.4. The tissue was then dissociated by trituration with fire-polished Pasteur and glass pipettes and cells were plated in petri dishes containing HBS and allowed to attach to the bottom of the dish for 1 or more hours before recordings. 2.2. Electrophysiology Petri dishes were mounted onto the stage of an inverted fluorescence microscope (IX70, Olympus) equipped with phase-contrast optics, and perfused (2– 4 ml/min) with HBS. Data were obtained with standard whole cell patch clamp techniques at room temperature (22–24°C) using borosilicate pipettes with final resistances of 4–6 M⍀ when filled with an intracellular solution of (in mM) CsCl 140, EGTA 10, HEPES 10, MgATP 4, Na-GTP 0.3 and CsOH for pH 7.3–7.4. After establishing whole-cell access, and to minimize the contribution of sodium and potassium currents, the perfusion medium was switched to a solution containing (in mM) TEA-Cl 150, CaCl2 5, glucose 10, HEPES 10, 1 µM tetrodotoxin and TEA-OH for pH 7.3–7.4. Media and drugs were applied with a gravity fed application system that consisted of a perfusion valve controller (VC6, Warner) and 200 µm tip (ALA) that generated a laminar flow. Solutions with conotoxins and agatoxin also contained cytochrome C (0.1%) to minimize peptide binding to containers. Lighting was subdued in some experiments to reduce drug degradation. Membrane currents were recorded with a patch-clamp amplifier (Axopatch 200B) and analyzed off-line with pCLAMP suite of programs (version 8). Linear components of capacitative and leak currents were subtracted using the standard P/4 protocol. Series resistance compensations of 70–80% were typically employed. If necessary, the rundown effects of channel blocking agents were compensated by using the linear regression of the current decrease. Cells with excessive or non-lin-
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ear rundown were excluded from this study. For statistical analyses we used SigmaStat. p values ⬍0.05 were consider significant. All values are reported as means±SEM. 2.3. Drugs ω-Conotoxin GVIA was obtained from Bachem and ω-agatoxin was obtained from Peptides International. Norepinephrine, clonidine, yohimbine, propranolol, UK14,304, phenylephrine, thermolysin, Mg-ATP, NaGTP and cytochrome C were obtained from Sigma and/or Sigma-RBI. Pronase was purchased from Calbiochem, TTX was purchased from Alomone and prazosin and nimodipine were purchased from Tocris.
3. Results 3.1. Properties of calcium currents in acutely dissociated MnPO neurons Data were obtained from 30 cells labeled with rhodamine fluorescent microspheres and considered to represent MnPO neurons whose axons projected to PVN. For comparison, we also evaluated responses from another 86 unlabeled neurons. As shown in Fig. 1 the non-inactivating inward current elicited by a 100 ms depolarizing step pulse starting from a VH of ⫺90 mV consisted of transient and steady state components, became evident at ⫺40 mV and achieved a peak of ⫺344±66 pA at ⫺10 mV (Fig. 1). The rise time (10– 90% at 0 mV) of the response was 2.2±0.2 ms. Responses were recognized as high voltage-activated calcium currents (HVA ICa) based on their reversal near +40 mV and sensitivity to cadmium (Fig. 1). Since observations from other 86 neurons that lacked any label yielded results (peak current of ⫺347±27 pA) that were not significantly different, the data were pooled. The relatively minor contribution from low voltage-activated calcium currents (⫺25.5±4.6 pA; n=10) was excluded from this analysis. 3.2. Effects of norepinephrine and adrenoceptor agonists and antagonists In the presence of norepinephrine, 80% of cells displayed a suppression of the HVA ICa current (Fig. 1) and prolongation in the rise time of response (to 2.6±0.2 ms; 119±4.4%; n=22; p⬍0.01). Responses were concentration dependent with peak current suppression of 17.3±2.7% (n=8) at 3 µM and 29.3±4.1% (n=6) at 30 µM. The norepinephrine response was significantly attenuated in the presence of yohimbine (3 µM), an α2-adrenoceptor antagonist (Fig. 2A and B; p⬍0.01), in sharp con-
trast with the lack of appreciable attenuation by either prazosin or propranolol, α1- and β-adrenoceptor antagonists, respectively (Fig. 2B). The effects of norepinephrine were mimicked by an α2-adrenoceptor agonist UK14,304, and to a lesser extent by clonidine whereas phenylephrine, an α1-adrenoceptor agonist, was only partially effective at the concentrations utilized (Fig. 2C and D) 3.3. Effects of G-protein modulation on norepinephrine-induced actions G proteins mediate the transmitter-mediated inhibition of HVA Ca2+ channels in a wide variety of neurons (Brown and Birnbaumer, 1990). GTP-γ-S, a nonhydrolyzable GTP analog that can render G-protein activation and channel modulation irreversible, has been reported to cause spontaneous reduction of Ca2+ current in the absence of applied agonist (Wollmuth et al., 1995). For this experiment we used 200 µM GTP-γ-S and 300 µM GTP in the pipette solution so as to achieve a turnover rate for GDP/GTP exchange that would minimize binding of the GTP-γ-S until norepinephrine could be applied. As illustrated in Fig. 3A–C, the response to the first drug application was almost irreversible, while a second application produced a significantly smaller effect (38±6.2 vs. 11.7±1.2%; p⬍0.05; n=3). We also tested the effects of NEM, a sulfhydryl-alkylating agent that can be applied acutely to inactivate the actions of the Go/Gi class of proteins and is reported to be most efficient at blocking G protein modulation that is sensitive to pertussis toxin (Shapiro et al., 1994). As illustrated in Fig. 3D–F, exposure to NEM (50 µM) for 4 min reduced the norepinephrine suppression from 27±5.1 to 7.2±1.3% (p⬍0.05, n=6). 3.4. NE inhibition of the Ca2+ current is partly voltage-dependent Some forms of the G protein-mediated neurotransmitter-induced suppression of HVA calcium currents express a voltage dependency, evident from their relief by strong depolarization (Hille, 1994). To test this possibility, we applied a double-pulse protocol (Fig. 4A) that could allow comparison between the number of channels available to open when initiated from a holding potential of ⫺80 mV to that when initiated after a depolarizing prepulse to +80 mV. As illustrated in Fig. 4A and B, the depolarizing prepulse reduced the response to norepinephrine from 35.3±3.9 to 19±3% (n=7; p⬍0.01) and to UK14,304 from 29.3±7.3 to 8.7±3.7% (n=3; p⬍0.05). In the presence of either drug the HVA ICa current during the first test pulse was diminished to a greater extent than during the pulse which followed strong depolarization, increasing the post/prepulse ratio
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Fig. 1. Norepinephrine suppresses HVA calcium currents. (A) On the left, sample current traces from another retrogradely-labeled MnPO neuron responding to voltage pulses (100 ms duration, applied in 10 mV increments) from a holding potential of ⫺80 mV. In the centre column, note the current suppression the presence of 50 µM norepinephrine (NE). On the right, calcium currents are blocked in a media containing 200 µM cadmium. (B) Current amplitudes at each potential measured at the peak of current responses (苲10 ms after start of depolarizing pulse) are plotted against corresponding membrane potential. Most acutely isolated MnPO neurons express only HVA calcium currents (HVA ICa) with no clear presence of LVA calcium currents. Note that cadmium blocks most of the current. (C) Representative current traces from the I/V relationship shown in B; numbers refer to stepping voltage. Bottom traces represent control, middle traces are in the presence of norepinephrine, top traces are in 200 µM cadmium.
to values near 1.2, consistent with a voltage-dependent component to this ability to suppress calcium currents. 3.5. HVA calcium channel subtypes in MnPO neurons and their suppression by norepinephrine Several channel subtypes contribute to HVA ICa currents in MnPO neurons. As illustrated in Fig. 5A and B, a percentage of this current was sensitive to each of the channel blocking agents applied. Results were independent of the order of application, indicating that the toxins each blocked distinct components at the concentrations used. Our analysis indicates that HVA currents in these dissociated neurons have a 40–45% contribution
from the N-type channels (sensitive to ω-CgTx), a 20– 25% contribution from L-type channels (sensitive to nimodipine) and a 15–20% contribution from P/Q type channels (sensitive to ω-AgaTx), with the remaining 10– 15% of the current as toxin insensitive and possibly mediated R-type channels. To determine the component(s) of calcium current, sensitive to norepinephrine, we tested its effects under control conditions and in the presence of the calcium channel antagonists. As illustrate in Fig. 6, only ω-CgTx significantly reduced suppression by norepinephrine (73.5±5% of the total drug-sensitive calcium current). Nimodipine and ω-AgaTx were insignificant blockers, affecting only a small portion of the drug-sensitive calcium currents (10.1±13.2 and 7.6±9.3%, respectively).
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Fig. 2. Norepinephrine responses are mediated by α2 adrenoceptors. (A) Representative traces illustrate the lack of effect of norepinephrine (20 µM) in the presence of yohimbine (3 µM). HVA ICa were evoked by stepping from a holding potential of ⫺80 to 0 mV every 20 s. (B) Pooled data for peak calcium currents from 9 cells illustrate a significant blockade of the response to norepinephrine (NE; 10–30 µM) by yohimbine (3 µM) but not by propranolol (3 µM) or prazosin (3 µM). (C) Individual traces from another neuron taken before and during each adrenoceptor agonist application (30 µM for 40 s). Note that the norepinephrine action is mimicked by UK14, 304 (UK) and to a lesser extent by clonidine (CLO), both α2 adrenoceptors agonists, whereas phenylephrine (PHE), an α1 adrenoceptor agonist, is marginally effective. (D) Pooled data show average peak calcium current suppression for all tested agonists. There was a significant difference between PHE effect and either NE or UK (p⬍0.05).
4. Discussion In-vivo studies, focused on the role of the lamina terminalis, have identified MnPO with an important role in maintaining salt and water balance (Mangiapane et al.,
1983; Gardiner et al., 1985; Johnson et al., 1996; McKinley et al., 1996). Anatomical tracer studies confirm that MnPO neurons are indeed connected with key sites involved in cardiovascular and autonomic functions (e.g. Saper and Levisohn, 1983). However, little is known about the intrinsic properties or neuropharmacology of MnPO neurons. In this analysis, we provided novel information of the characteristics of their HVA ICa, and the differential contributions from L, N and P/Q type channels. In addition, we demonstrated that norepinephrine, featured prominently among the afferents to MnPO, can act via α2 adrenoceptors to suppress these HVACa currents, largely through an action on N-type channels. Norepinephrine is known to modulate calcium channels in a variety of cells, an action that involves G-proteins, either directly or through second messengers (Anwyl, 1991; Ikeda 1992, 1996; Hille, 1994; Dolphin, 1998), often of the Go/Gi class that are sensitive to pertussis toxin (Milligan, 1988). PTX-sensitive G-proteins can be uncoupled from their receptors by N-ethylmaleimide (NEM) a sulfhydryl alkylating agent (Nakajima et al., 1990; Shapiro et al., 1994). The norepinephrine suppression of calcium currents in MnPO neurons is enhanced by pretreatment with GTP-γ-S, an activator of G-proteins. Blockade of the effect by NEM suggests mediation through PTX-sensitive G-proteins. Norepinephrine acts via α2 adrenoceptors to slow the activation kinetics of calcium channels. Norepinephrine’s effects can be partially relieved by large depolarizing prepulses, indicating a voltage-dependent component (Bean, 1989; Hille, 1994). This resembles norepinephrine’s effects on calcium currents in caudal raphe neurons (Li and Bayliss, 1998) and is most often seen where calcium currents are suppressed by neurotransmitters acting via PTX-sensitive G-proteins (Hille, 1994). Notable exceptions include an action in rat superior cervical ganglion cells by a voltage-dependent pathway that is PTXinsensitive (Bernheim et al., 1991; Zhu and Ikeda, 1994), and in chick dorsal root ganglia neurons by PTX sensitive voltage-independent pathway (Diverse-Pierluissi et al., 1995). Although not directly tested, the voltage dependence of inhibition seen here may reflect direct binding of a G-protein, probably the Gβγ heterodimer, to calcium channels through a membrane-delimited transduction mechanism (Herlitze et al., 1996; Ikeda, 1996). In this study we used nimodipine at 10 µM to block L type HVACa channels. In some preparations this concentration can partially block conotoxin-sensitive channels (Churchill and MacVicar, 1994). However, in our preparation the conotoxin effect alone was not significantly different from that in this concentration of nimodipine (Fig. 5B). In this first report on HVACa channels, we noted that N-type channels carry a substantial component (40– 45%) of the calcium current. These are also the channels
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Fig. 3. G-proteins participate in the norepinephrine-induced suppression of HVA ICa. (A) Pooled data from three MnPO neurons loaded with GTP-γ-S plots the calcium current (peak component) as a percentage of control (average of first three responses) vs. time. Application of norepinephrine (NE, 50 µM) induces an irreversible suppression of the current. (B) Sample traces from a neuron used in A. (C) Histogram illustrates that the response to a second application of norepinephrine is significantly reduced (p⬍0.05). (D) Plot to illustrate NEM (50 µM) blockade of the norepinephrine suppression of peak currents. (E). Sample traces from the neuron shown in D. (F) Pooled data from six neurons illustrate a significant attenuation (p⬍0.05) of the suppressant action of norepinephrine (30 µM) after exposure to NEM.
Fig. 4. Evidence for a voltage-dependent component of the α2 adrenoceptor effect. (A) The inset indicates the voltage pulse protocol used to assess voltage dependency: an initial test pulse to 0 mV before (pre) and then following (post) a large depolarizing step to +80 mV. Illustrated are the actual current responses for each trial with norepinephrine (NE; 30 µM) or UK14,304 (UK; 30 µM). Note the reduced response to each drug in the post trial after the strong depolarization. (B) Summary histogram indicates the significant reduction on peak amplitude of the suppressant actions of both norepinephrine (p⬍0.01) and UK14,304 (p⬍0.05) after the conditioning depolarizing step.
most affected by norepinephrine. Such a differential expression of neurotransmitter suppression of calcium currents is not unique. For example, norepinephrine is reported to block N-type calcium currents selectively in frog sympathetic neurons (Boland et al., 1994) and rabbit parasympathetic neurons (Akasu et al., 1990). As well, suppression of both N- and P/Q-type channels can follow activation of GABAB (Harayama et al., 1998), cholinergic muscarinic (Yan et al., 1997) and dopamine receptors (Surmeier et al., 1995). In general, a greater fraction of N- rather than P/Q-subtype channels are the targets for this suppression (Amico et al., 1995; Ishibashi and Akaike, 1995a), although results vary among cells and receptor subtypes. For instance, rat hippocampal neurons express both N- and P/Q-type channels but only the N-type is modulated by somatostatin (Ishibashi and Akaike, 1995b); in CA3 neurons, adenosine A1 receptors act to suppress calcium currents via effects on Ntype channels whereas A2 receptors enhance calcium currents via an action on P/Q-type channels (Mogul et al., 1993). Although patch clamp recordings from MnPO neurons in slice preparations commonly display low threshold spikes and rebound depolarizations (Bai and Renaud, 1998), the dissociated MnPO neurons observed in this study presented with marginal LVACa currents. The dissociation procedure necessarily results in a loss of den-
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Fig. 5. MnPO neurons possess multiple subtypes of HVA ICa. (A) Peak amplitudes of calcium currents were measured during voltage steps from a holding potential of ⫺80 to 0 mV and plotted as a function of time. P/Q type blocker ω-agatoxin IVA (AGA; 100 nM), N-type blocker ω-conotoxin GVIA (CTX; 2 µM) and L-type blocker nimodipine (NIM; 10 µM) were applied at the times indicated by horizontal bars. Inset: calcium channel currents evoked under indicated conditions. (B) Summary data show effects of three different calciumchannel blockers on the peak component of HVACa currents in MnPO neurons. These data suggest that the most abundant type of HVA calcium channel in these neurons are N-type (40–45%) followed by Ltype (20–25%), P/Q-type (15–20%) and the rest is toxin insensitive Rtype (estimated 10–25%).
Fig. 6. NE inhibits predominantly N-type calcium currents. Individual traces on the left show calcium currents evoked before and during NE application (left column). Middle column shows traces evoked before and during treatment with different calcium channel blockers (A, nimodipine 10 µM, NIM; B, ω-conotoxin GVIA, 1 µM, CTX; C, ω-agatoxin IVA, 100 nM, AGA) and during combined application of NE plus channel blocker. Pooled data are displayed in histograms on the right. Note that only CTX significantly reduces norepinephrineinduced inhibition (p⬍0.001). Number of tested cells: NIM=6, CTX=4 and AGA=6.
dritic processes, and the loss of response may indicate a dendritic location for the majority of these LVA calcium channels (c.f. Mouginot et al., 1997). A norepinephrine innervation to MnPO is vital to its function. Witness the profound disruption in hydromineral homeostasis that follows depletion of its catecholaminergic innervation by 6-hydroxydopamine, and the functional restoration by norepinephrine (Cunningham and Johnson 1989, 1991). Until recently, little was known of norepinephrine’s actions in MnPO at the cellular level. Results with in-vivo extracellular recordings from MnPO following electrical stimulation in the medullary catecholamine cell groups suggested that this input was excitatory and involved α adrenoceptors (Tanaka et al., 1992). By contrast, in a recent in-
vitro patch clamp analysis (Bai and Renaud, 1998) we noted that exogenously applied norepinephrine induced two patterns of postsynaptic response, both involving potassium channels: a majority (苲60%) of responsive cells revealed an α2 adrenoceptor-mediated membrane hyperpolarization; a minority revealed an α1-adrenoceptor-mediated depolarization. The present observations now indicate that postsynaptic α2 adrenoceptors can also modulate (inhibit) N-type calcium channels, and the higher percentage of responsive cells suggests that this is widespread and likely to affect a majority of MnPO neurons. More investigation is required as to how this action of norepinephrine is translated into altered cell excitability, e.g. through an influence on calcium-activated potassium channels.
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Acknowledgements Supported by the Canadian Institutes for Health Research and the Heart and Stroke Foundation of Canada.
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Norepinephrine acts via α2 adrenergic receptors to suppress N-type calcium channels in dissociated rat median preoptic nucleus neurons M. Kolaj, L.P. Renaud
*
Neurology and Neurosciences, Loeb Health Research Institute, Ottawa Hospital —- Civic Site and University of Ottawa, 1053 Carling Avenue, Ottawa, Ont., Canada K1Y 4E9 Received 2 April 2001; received in revised form 25 April 2001; accepted 14 June 2001
Abstract The median preoptic (MnPO) nucleus, a key CNS site for hydromineral and cardiovascular homeostasis, receives a dense norepinephrine innervation from brainstem autonomic centers. Since norepinephrine is known to influence neuronal excitability by modulating calcium channel function, we applied whole cell patch clamp techniques to study calcium currents in 116 dissociated MnPO neurons, including 30 cells identified by a retrograde label as projecting to the hypothalamic paraventricular nucleus. Norepinephrine (3–50 µM) suppressed high-voltage-activated calcium currents (HVA ICa) in 80% of cells, selectively blockable by yohimbine and mimicked by UK14,304 and clonidine. The norepinephrine effect was relieved by strong prior depolarization, indicating a voltagedependent component. Intracellular GTP-γ-S blocked the effect. Blockade by extracellular NEM suggested involvement of pertussistoxin sensitive G-proteins. Based on pharmacological properties, these HVA ICas had the following composition: 40–45% N-type (blockable by ω-conotoxin GVIA); 20–25% L-type (blockable by nimodipine); 15–20% P/Q-type (blockable by ω-agatoxin IVA). Since 苲75% of the norepinephrine effect was blockable with ω-conotoxin GVIA, we conclude that postsynaptic α2 adrenoceptors preferentially suppress N-type calcium channels, revealing a novel mechanism whereby norepinephrine can modulate excitability in MnPO neurons. 2001 Elsevier Science Ltd. All rights reserved. Keywords: High voltage calcium currents; N type channels; Median preoptic nucleus; Norepinephrine; Dissociated cells; Patch clamp technique
1. Introduction Calcium is an important participant in the control of many neuronal functions, including regulation of their excitability, and voltage-dependent Ca2+ channels provide one of the many routes of calcium entry into neurons. Initially these channels were classified as low- and high-voltage-activated (LVA and HVA) currents on the basis of their differential activation and inactivation properties (Carbone and Swadulla, 1993). Multiple subtypes of HVA calcium currents (HVA ICa) can now be identified on the basis of their electrophysiological and pharmacological characteristics: nimodipine- or dihydropyridine-sensitive L-type currents, ω-conotoxin GVIA-
* Corresponding author. Tel.: +1-613-761-5070; fax: +1-613-7615360. E-mail address: [email protected] (L.P. Renaud).
sensitive N type currents, ω-agatoxin IVA-sensitive P/Q type currents, and a residual R-type that is insensitive to channel antagonists. Importantly, a wide variety of neurotransmitters and peptides, including norepinephrine, can be shown to modulate (usually inhibit) voltage-dependent Ca2+ channels (Brown and Birnbaumer, 1990; Anwyl, 1991; Hille, 1994). The characterization of calcium currents and their modulation are important for understanding the function of central neurons, including those comprising the median preoptic nucleus (MnPO), a parvocellular group of cells located at the midpoint of the lamina terminalis forming the anterior wall of the third cerebral ventricle. A variety of whole animal studies attest to the importance of the MnPO in achieving behavioral and endocrine responses to intracerebroventricular (icv) angiotensin, and in maintaining hydromineral homeostasis (Johnson et al., 1996; McKinley et al., 1996). Lesions of the MnPO severely disrupt salt and water balance,
0028-3908/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 1 ) 0 0 0 9 0 - 9
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cardiovascular and behavioral responses to icv angiotensin (Mangiapane et al., 1983; Gardiner et al., 1985; Cunningham and Johnson, 1989; Xu and Hebert, 1995). There is reason to suspect that the brainstem innervation to this region is also critical to normal function. MnPO is the recipient of a prominent catecholaminergic (i.e. norepinephrine) input arising from the medulla (Saper and Levisohn, 1983; Kawano and Masuko, 1993). Neurotoxin-induced lesions of these catecholaminergic fibers markedly disrupt both hydromineral balance and angiotensin-induced drinking behavior, events that can be restored with norepinephrinergic transplants or infusions (McRae-Degueurce et al., 1986; Cunningham and Johnson 1989, 1991). Interestingly, similar deficits follow icv pretreatment with HVACa channel antagonists (Zhu and Herbert, 1997). However little is known of the neuronal mechanisms operative within this nucleus, including the intrinsic cellular properties and the neuropharmacology of its constitutive neurons. As an initial approach, we have used whole cell patch clamp technique in brain slices to begin a characterization of the intrinsic properties unique to MnPO neurons, and to evaluate their transmitter receptors. In a recent analysis (Bai and Renaud, 1998), we noted that 苲60% of neurons responded to bath applied norepinephrine with a postsynaptic α2-mediated membrane hyperpolarization or an α1-mediated membrane depolarization, both likely involving changes in potassium ionic conductances (Bai and Renaud, 1998). To extend this analysis, we sought to focus on MnPO neurons identified as projecting their axons to a known neuroendocrine and autonomic target of MnPO neurons i.e. the hypothalamic paraventricular nucleus or PVN (Silverman et al., 1981). Taking advantage of retrograde labeling from PVN and the resolution of dissociated cell preparations of MnPO, we have addressed the hypothesis that norepinephrine could also have a modulating influence on their calcium currents. We now report that L, N and P/Q type channels contribute to HVA ICa in MnPO neurons, and that norepinephrine selectively inhibits their N-type channels. A preliminary account of these results has been presented (Kolaj et al., 2000).
2. Methods 2.1. Preparation Adult male Long–Evans rats (60–120 g body wt) were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Animals were initially anesthetized with Somnotol (50 mg/kg), placed in a stereotaxic frame and a burr hole drilled on each side of midline to allow insertion of a micropipette. Animals received 0.2 µl of a suspension of a retrograde tracer (rhodamine latex microspheres; Molecular Probes Inc.) delivered by stereotaxic microinjection over 8–
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10 min into each hypothalamic paraventricular nucleus (coordinates from bregma: ⫺1.0, lateral 0.5, depth 6.5– 7.0). After closure of the skin with nylon sutures, animals were returned to individual cages. About two to seven days later, animals were sacrificed by decapitation, the brain quickly removed and placed in cooled (4°C) gassed (O2) PIPES-buffered solution (PBS, in mM: NaCl 120, KCl 5, glucose 25, CaCl2 1, MgCl2 1, PIPES 20 and NaOH for pH 7.1). The brain was blocked and sectioned at 250–300 µm with a vibratome, retaining the section that contained the MnPO and anterior commissure. The MnPO area was punched out and placed in an oxygenated bathing solution (PBS) containing pronase (0.1 mg/ml, Calbiochem) for 15 min. After transfer to a solution containing thermolysin (0.1 mg/ml, Sigma) for 15 min at 35°C, the tissue section was rinsed in PBS, then washed several times in a HEPES-buffered solution (HBS) containing (in mM) NaCl 150, KCl 5, glucose 10, CaCl2 2, MgCl2 1, Hepes 10 at pH 7.3–7.4. The tissue was then dissociated by trituration with fire-polished Pasteur and glass pipettes and cells were plated in petri dishes containing HBS and allowed to attach to the bottom of the dish for 1 or more hours before recordings. 2.2. Electrophysiology Petri dishes were mounted onto the stage of an inverted fluorescence microscope (IX70, Olympus) equipped with phase-contrast optics, and perfused (2– 4 ml/min) with HBS. Data were obtained with standard whole cell patch clamp techniques at room temperature (22–24°C) using borosilicate pipettes with final resistances of 4–6 M⍀ when filled with an intracellular solution of (in mM) CsCl 140, EGTA 10, HEPES 10, MgATP 4, Na-GTP 0.3 and CsOH for pH 7.3–7.4. After establishing whole-cell access, and to minimize the contribution of sodium and potassium currents, the perfusion medium was switched to a solution containing (in mM) TEA-Cl 150, CaCl2 5, glucose 10, HEPES 10, 1 µM tetrodotoxin and TEA-OH for pH 7.3–7.4. Media and drugs were applied with a gravity fed application system that consisted of a perfusion valve controller (VC6, Warner) and 200 µm tip (ALA) that generated a laminar flow. Solutions with conotoxins and agatoxin also contained cytochrome C (0.1%) to minimize peptide binding to containers. Lighting was subdued in some experiments to reduce drug degradation. Membrane currents were recorded with a patch-clamp amplifier (Axopatch 200B) and analyzed off-line with pCLAMP suite of programs (version 8). Linear components of capacitative and leak currents were subtracted using the standard P/4 protocol. Series resistance compensations of 70–80% were typically employed. If necessary, the rundown effects of channel blocking agents were compensated by using the linear regression of the current decrease. Cells with excessive or non-lin-
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ear rundown were excluded from this study. For statistical analyses we used SigmaStat. p values ⬍0.05 were consider significant. All values are reported as means±SEM. 2.3. Drugs ω-Conotoxin GVIA was obtained from Bachem and ω-agatoxin was obtained from Peptides International. Norepinephrine, clonidine, yohimbine, propranolol, UK14,304, phenylephrine, thermolysin, Mg-ATP, NaGTP and cytochrome C were obtained from Sigma and/or Sigma-RBI. Pronase was purchased from Calbiochem, TTX was purchased from Alomone and prazosin and nimodipine were purchased from Tocris.
3. Results 3.1. Properties of calcium currents in acutely dissociated MnPO neurons Data were obtained from 30 cells labeled with rhodamine fluorescent microspheres and considered to represent MnPO neurons whose axons projected to PVN. For comparison, we also evaluated responses from another 86 unlabeled neurons. As shown in Fig. 1 the non-inactivating inward current elicited by a 100 ms depolarizing step pulse starting from a VH of ⫺90 mV consisted of transient and steady state components, became evident at ⫺40 mV and achieved a peak of ⫺344±66 pA at ⫺10 mV (Fig. 1). The rise time (10– 90% at 0 mV) of the response was 2.2±0.2 ms. Responses were recognized as high voltage-activated calcium currents (HVA ICa) based on their reversal near +40 mV and sensitivity to cadmium (Fig. 1). Since observations from other 86 neurons that lacked any label yielded results (peak current of ⫺347±27 pA) that were not significantly different, the data were pooled. The relatively minor contribution from low voltage-activated calcium currents (⫺25.5±4.6 pA; n=10) was excluded from this analysis. 3.2. Effects of norepinephrine and adrenoceptor agonists and antagonists In the presence of norepinephrine, 80% of cells displayed a suppression of the HVA ICa current (Fig. 1) and prolongation in the rise time of response (to 2.6±0.2 ms; 119±4.4%; n=22; p⬍0.01). Responses were concentration dependent with peak current suppression of 17.3±2.7% (n=8) at 3 µM and 29.3±4.1% (n=6) at 30 µM. The norepinephrine response was significantly attenuated in the presence of yohimbine (3 µM), an α2-adrenoceptor antagonist (Fig. 2A and B; p⬍0.01), in sharp con-
trast with the lack of appreciable attenuation by either prazosin or propranolol, α1- and β-adrenoceptor antagonists, respectively (Fig. 2B). The effects of norepinephrine were mimicked by an α2-adrenoceptor agonist UK14,304, and to a lesser extent by clonidine whereas phenylephrine, an α1-adrenoceptor agonist, was only partially effective at the concentrations utilized (Fig. 2C and D) 3.3. Effects of G-protein modulation on norepinephrine-induced actions G proteins mediate the transmitter-mediated inhibition of HVA Ca2+ channels in a wide variety of neurons (Brown and Birnbaumer, 1990). GTP-γ-S, a nonhydrolyzable GTP analog that can render G-protein activation and channel modulation irreversible, has been reported to cause spontaneous reduction of Ca2+ current in the absence of applied agonist (Wollmuth et al., 1995). For this experiment we used 200 µM GTP-γ-S and 300 µM GTP in the pipette solution so as to achieve a turnover rate for GDP/GTP exchange that would minimize binding of the GTP-γ-S until norepinephrine could be applied. As illustrated in Fig. 3A–C, the response to the first drug application was almost irreversible, while a second application produced a significantly smaller effect (38±6.2 vs. 11.7±1.2%; p⬍0.05; n=3). We also tested the effects of NEM, a sulfhydryl-alkylating agent that can be applied acutely to inactivate the actions of the Go/Gi class of proteins and is reported to be most efficient at blocking G protein modulation that is sensitive to pertussis toxin (Shapiro et al., 1994). As illustrated in Fig. 3D–F, exposure to NEM (50 µM) for 4 min reduced the norepinephrine suppression from 27±5.1 to 7.2±1.3% (p⬍0.05, n=6). 3.4. NE inhibition of the Ca2+ current is partly voltage-dependent Some forms of the G protein-mediated neurotransmitter-induced suppression of HVA calcium currents express a voltage dependency, evident from their relief by strong depolarization (Hille, 1994). To test this possibility, we applied a double-pulse protocol (Fig. 4A) that could allow comparison between the number of channels available to open when initiated from a holding potential of ⫺80 mV to that when initiated after a depolarizing prepulse to +80 mV. As illustrated in Fig. 4A and B, the depolarizing prepulse reduced the response to norepinephrine from 35.3±3.9 to 19±3% (n=7; p⬍0.01) and to UK14,304 from 29.3±7.3 to 8.7±3.7% (n=3; p⬍0.05). In the presence of either drug the HVA ICa current during the first test pulse was diminished to a greater extent than during the pulse which followed strong depolarization, increasing the post/prepulse ratio
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Fig. 1. Norepinephrine suppresses HVA calcium currents. (A) On the left, sample current traces from another retrogradely-labeled MnPO neuron responding to voltage pulses (100 ms duration, applied in 10 mV increments) from a holding potential of ⫺80 mV. In the centre column, note the current suppression the presence of 50 µM norepinephrine (NE). On the right, calcium currents are blocked in a media containing 200 µM cadmium. (B) Current amplitudes at each potential measured at the peak of current responses (苲10 ms after start of depolarizing pulse) are plotted against corresponding membrane potential. Most acutely isolated MnPO neurons express only HVA calcium currents (HVA ICa) with no clear presence of LVA calcium currents. Note that cadmium blocks most of the current. (C) Representative current traces from the I/V relationship shown in B; numbers refer to stepping voltage. Bottom traces represent control, middle traces are in the presence of norepinephrine, top traces are in 200 µM cadmium.
to values near 1.2, consistent with a voltage-dependent component to this ability to suppress calcium currents. 3.5. HVA calcium channel subtypes in MnPO neurons and their suppression by norepinephrine Several channel subtypes contribute to HVA ICa currents in MnPO neurons. As illustrated in Fig. 5A and B, a percentage of this current was sensitive to each of the channel blocking agents applied. Results were independent of the order of application, indicating that the toxins each blocked distinct components at the concentrations used. Our analysis indicates that HVA currents in these dissociated neurons have a 40–45% contribution
from the N-type channels (sensitive to ω-CgTx), a 20– 25% contribution from L-type channels (sensitive to nimodipine) and a 15–20% contribution from P/Q type channels (sensitive to ω-AgaTx), with the remaining 10– 15% of the current as toxin insensitive and possibly mediated R-type channels. To determine the component(s) of calcium current, sensitive to norepinephrine, we tested its effects under control conditions and in the presence of the calcium channel antagonists. As illustrate in Fig. 6, only ω-CgTx significantly reduced suppression by norepinephrine (73.5±5% of the total drug-sensitive calcium current). Nimodipine and ω-AgaTx were insignificant blockers, affecting only a small portion of the drug-sensitive calcium currents (10.1±13.2 and 7.6±9.3%, respectively).
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Fig. 2. Norepinephrine responses are mediated by α2 adrenoceptors. (A) Representative traces illustrate the lack of effect of norepinephrine (20 µM) in the presence of yohimbine (3 µM). HVA ICa were evoked by stepping from a holding potential of ⫺80 to 0 mV every 20 s. (B) Pooled data for peak calcium currents from 9 cells illustrate a significant blockade of the response to norepinephrine (NE; 10–30 µM) by yohimbine (3 µM) but not by propranolol (3 µM) or prazosin (3 µM). (C) Individual traces from another neuron taken before and during each adrenoceptor agonist application (30 µM for 40 s). Note that the norepinephrine action is mimicked by UK14, 304 (UK) and to a lesser extent by clonidine (CLO), both α2 adrenoceptors agonists, whereas phenylephrine (PHE), an α1 adrenoceptor agonist, is marginally effective. (D) Pooled data show average peak calcium current suppression for all tested agonists. There was a significant difference between PHE effect and either NE or UK (p⬍0.05).
4. Discussion In-vivo studies, focused on the role of the lamina terminalis, have identified MnPO with an important role in maintaining salt and water balance (Mangiapane et al.,
1983; Gardiner et al., 1985; Johnson et al., 1996; McKinley et al., 1996). Anatomical tracer studies confirm that MnPO neurons are indeed connected with key sites involved in cardiovascular and autonomic functions (e.g. Saper and Levisohn, 1983). However, little is known about the intrinsic properties or neuropharmacology of MnPO neurons. In this analysis, we provided novel information of the characteristics of their HVA ICa, and the differential contributions from L, N and P/Q type channels. In addition, we demonstrated that norepinephrine, featured prominently among the afferents to MnPO, can act via α2 adrenoceptors to suppress these HVACa currents, largely through an action on N-type channels. Norepinephrine is known to modulate calcium channels in a variety of cells, an action that involves G-proteins, either directly or through second messengers (Anwyl, 1991; Ikeda 1992, 1996; Hille, 1994; Dolphin, 1998), often of the Go/Gi class that are sensitive to pertussis toxin (Milligan, 1988). PTX-sensitive G-proteins can be uncoupled from their receptors by N-ethylmaleimide (NEM) a sulfhydryl alkylating agent (Nakajima et al., 1990; Shapiro et al., 1994). The norepinephrine suppression of calcium currents in MnPO neurons is enhanced by pretreatment with GTP-γ-S, an activator of G-proteins. Blockade of the effect by NEM suggests mediation through PTX-sensitive G-proteins. Norepinephrine acts via α2 adrenoceptors to slow the activation kinetics of calcium channels. Norepinephrine’s effects can be partially relieved by large depolarizing prepulses, indicating a voltage-dependent component (Bean, 1989; Hille, 1994). This resembles norepinephrine’s effects on calcium currents in caudal raphe neurons (Li and Bayliss, 1998) and is most often seen where calcium currents are suppressed by neurotransmitters acting via PTX-sensitive G-proteins (Hille, 1994). Notable exceptions include an action in rat superior cervical ganglion cells by a voltage-dependent pathway that is PTXinsensitive (Bernheim et al., 1991; Zhu and Ikeda, 1994), and in chick dorsal root ganglia neurons by PTX sensitive voltage-independent pathway (Diverse-Pierluissi et al., 1995). Although not directly tested, the voltage dependence of inhibition seen here may reflect direct binding of a G-protein, probably the Gβγ heterodimer, to calcium channels through a membrane-delimited transduction mechanism (Herlitze et al., 1996; Ikeda, 1996). In this study we used nimodipine at 10 µM to block L type HVACa channels. In some preparations this concentration can partially block conotoxin-sensitive channels (Churchill and MacVicar, 1994). However, in our preparation the conotoxin effect alone was not significantly different from that in this concentration of nimodipine (Fig. 5B). In this first report on HVACa channels, we noted that N-type channels carry a substantial component (40– 45%) of the calcium current. These are also the channels
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Fig. 3. G-proteins participate in the norepinephrine-induced suppression of HVA ICa. (A) Pooled data from three MnPO neurons loaded with GTP-γ-S plots the calcium current (peak component) as a percentage of control (average of first three responses) vs. time. Application of norepinephrine (NE, 50 µM) induces an irreversible suppression of the current. (B) Sample traces from a neuron used in A. (C) Histogram illustrates that the response to a second application of norepinephrine is significantly reduced (p⬍0.05). (D) Plot to illustrate NEM (50 µM) blockade of the norepinephrine suppression of peak currents. (E). Sample traces from the neuron shown in D. (F) Pooled data from six neurons illustrate a significant attenuation (p⬍0.05) of the suppressant action of norepinephrine (30 µM) after exposure to NEM.
Fig. 4. Evidence for a voltage-dependent component of the α2 adrenoceptor effect. (A) The inset indicates the voltage pulse protocol used to assess voltage dependency: an initial test pulse to 0 mV before (pre) and then following (post) a large depolarizing step to +80 mV. Illustrated are the actual current responses for each trial with norepinephrine (NE; 30 µM) or UK14,304 (UK; 30 µM). Note the reduced response to each drug in the post trial after the strong depolarization. (B) Summary histogram indicates the significant reduction on peak amplitude of the suppressant actions of both norepinephrine (p⬍0.01) and UK14,304 (p⬍0.05) after the conditioning depolarizing step.
most affected by norepinephrine. Such a differential expression of neurotransmitter suppression of calcium currents is not unique. For example, norepinephrine is reported to block N-type calcium currents selectively in frog sympathetic neurons (Boland et al., 1994) and rabbit parasympathetic neurons (Akasu et al., 1990). As well, suppression of both N- and P/Q-type channels can follow activation of GABAB (Harayama et al., 1998), cholinergic muscarinic (Yan et al., 1997) and dopamine receptors (Surmeier et al., 1995). In general, a greater fraction of N- rather than P/Q-subtype channels are the targets for this suppression (Amico et al., 1995; Ishibashi and Akaike, 1995a), although results vary among cells and receptor subtypes. For instance, rat hippocampal neurons express both N- and P/Q-type channels but only the N-type is modulated by somatostatin (Ishibashi and Akaike, 1995b); in CA3 neurons, adenosine A1 receptors act to suppress calcium currents via effects on Ntype channels whereas A2 receptors enhance calcium currents via an action on P/Q-type channels (Mogul et al., 1993). Although patch clamp recordings from MnPO neurons in slice preparations commonly display low threshold spikes and rebound depolarizations (Bai and Renaud, 1998), the dissociated MnPO neurons observed in this study presented with marginal LVACa currents. The dissociation procedure necessarily results in a loss of den-
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Fig. 5. MnPO neurons possess multiple subtypes of HVA ICa. (A) Peak amplitudes of calcium currents were measured during voltage steps from a holding potential of ⫺80 to 0 mV and plotted as a function of time. P/Q type blocker ω-agatoxin IVA (AGA; 100 nM), N-type blocker ω-conotoxin GVIA (CTX; 2 µM) and L-type blocker nimodipine (NIM; 10 µM) were applied at the times indicated by horizontal bars. Inset: calcium channel currents evoked under indicated conditions. (B) Summary data show effects of three different calciumchannel blockers on the peak component of HVACa currents in MnPO neurons. These data suggest that the most abundant type of HVA calcium channel in these neurons are N-type (40–45%) followed by Ltype (20–25%), P/Q-type (15–20%) and the rest is toxin insensitive Rtype (estimated 10–25%).
Fig. 6. NE inhibits predominantly N-type calcium currents. Individual traces on the left show calcium currents evoked before and during NE application (left column). Middle column shows traces evoked before and during treatment with different calcium channel blockers (A, nimodipine 10 µM, NIM; B, ω-conotoxin GVIA, 1 µM, CTX; C, ω-agatoxin IVA, 100 nM, AGA) and during combined application of NE plus channel blocker. Pooled data are displayed in histograms on the right. Note that only CTX significantly reduces norepinephrineinduced inhibition (p⬍0.001). Number of tested cells: NIM=6, CTX=4 and AGA=6.
dritic processes, and the loss of response may indicate a dendritic location for the majority of these LVA calcium channels (c.f. Mouginot et al., 1997). A norepinephrine innervation to MnPO is vital to its function. Witness the profound disruption in hydromineral homeostasis that follows depletion of its catecholaminergic innervation by 6-hydroxydopamine, and the functional restoration by norepinephrine (Cunningham and Johnson 1989, 1991). Until recently, little was known of norepinephrine’s actions in MnPO at the cellular level. Results with in-vivo extracellular recordings from MnPO following electrical stimulation in the medullary catecholamine cell groups suggested that this input was excitatory and involved α adrenoceptors (Tanaka et al., 1992). By contrast, in a recent in-
vitro patch clamp analysis (Bai and Renaud, 1998) we noted that exogenously applied norepinephrine induced two patterns of postsynaptic response, both involving potassium channels: a majority (苲60%) of responsive cells revealed an α2 adrenoceptor-mediated membrane hyperpolarization; a minority revealed an α1-adrenoceptor-mediated depolarization. The present observations now indicate that postsynaptic α2 adrenoceptors can also modulate (inhibit) N-type calcium channels, and the higher percentage of responsive cells suggests that this is widespread and likely to affect a majority of MnPO neurons. More investigation is required as to how this action of norepinephrine is translated into altered cell excitability, e.g. through an influence on calcium-activated potassium channels.
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Acknowledgements Supported by the Canadian Institutes for Health Research and the Heart and Stroke Foundation of Canada.
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