Functional properties of dopaminergic neurones in the mouse olfactory bulb
1 Università di Ferrara, Dip. Biologia, Sezione di Fisiologia e Biofisica - Centro di Neuroscienze, Via Borsari, 46-44100 Ferrara, Italy
2 Fukushima Medical University School of Medicine, Department Molecular Genetics, Hikarigaoka, Fukushima 960-1295, Japan
3 Keio University, School of Medicine, Department of Physiology, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Abstract
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The olfactory bulb of mammals contains a large population of dopaminergic interneurones within the glomerular layer. Dopamine has been shown both in vivo and in vitro to modulate several aspects of olfactory information processing, but the functional properties of dopaminergic neurones have never been described due to the inability to recognize these cells in living preparations. To overcome this difficulty, we used a transgenic mouse strain harbouring an eGFP (enhanced green fluorescent protein) reporter construct under the promoter of tyrosine hydroxylase, the rate-limiting enzyme for cathecolamine synthesis. As a result, we were able to identify dopaminergic neurones (TH-GFP cells) in living preparations and, for the first time, we could study the functional properties of such neurones in the olfactory bulb, in both slices and dissociated cells. The most prominent feature of these cells was the autorhythmicity. In these cells we identified five main voltage-dependent conductances: the two having largest amplitude were a fast transient Na+ current and a delayed rectifier K+ current. In addition, we observed three smaller inward currents, sustained by Na+ ions (persistent type) and by Ca2+ ions (LVA and HVA). Using pharmacological tools and ion substitution methods we showed that the pacemaking process is supported by the interplay of the persistent Na+ current and of a T-type Ca2+ current. We carried out a complete kinetical analysis of the five conductances present in these cells, and developed a Hodgkin-Huxley model of TH-GFP cells, capable of reproducing accurately the properties of living cells, including autorhytmicity, and allowing a precise understanding of the process.
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Introduction
In the mammalian olfactory bulb (OB) dopaminergic (DA) neurones constitute a fraction of the cells occupying the most external (glomerular) layer (Halász et al. 1977). In this region, populated by three types of interneurones, periglomerular (PG) cells, short-axon cells and external tufted (ET) cells (Halász, 1990) – often collectively referred to as juxtaglomerular cells – an estimated 10% of the neurones in adulthood are positive for tyrosine hydroxylase (TH) (McLean & Shipley, 1988; Kratskin & Belluzzi, 2003), the rate-limiting enzyme for dopamine synthesis. Dopaminergic neurones in the glomerular layer include PG cells (Kosaka et al. 1985; Gall et al. 1987) and a fraction of ET cells (Halász, 1990). Several studies have focused on the role of dopamine in the olfactory bulb, using immunohistochemical (Baker et al. 1983; Guthrie et al. 1991), behavioural (Doty & Risser, 1989) and electrophysiological techniques (Nowycky et al. 1983; Ennis et al. 2001; Davila et al. 2003). Despite the wealth of information available on the role of dopamine in olfaction, the functional properties of DA neurones in the OB remains entirely unknown.
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A property shared by many DA neurones in the CNS is their capacity to generate rhythmic action potentials even in the absence of synaptic inputs (Grace & Onn, 1989; Hainsworth et al. 1991; Yung et al. 1991; Feigenspan et al. 1998; Neuhoff et al. 2002). In this paper we show for the first time that DA cells in the glomerular layer of the olfactory bulb possess a pacemaker activity, and we provide an explanation for the ionic basis of rhythm generation in these cells.
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There is an additional reason to study the functional properties of DA neurones in the OB other than their role in olfaction. The olfactory bulb is one of the rare regions of the mammalian CNS in which new cells, derived from stem cells in the anterior subventricular zone, are also added in adulthood (Gross, 2000). In the OB, these cells differentiate in interneurones in the granular and glomerular layers. Among these cells there are DA neurones (Betarbet et al. 1996; Baker et al. 2001), and this has raised a remarkable interest because, with their accessibility, they could provide a convenient source of autologous DA neurones for transplant therapies in neurodegenerative diseases, like Parkinson's disease.
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Electrical recordings from identified DA neurones have been largely impeded by the difficulty of discerning these cells in living brain tissue. Our experimental approach to specifically target DA neurones for electrophysiological analysis was to use transgenic mice carrying green fluorescent protein (GFP) under the control of TH promoter (TH-GFP cells) (Sawamoto et al. 2001; Matsushita et al. 2002), thereby tagging live DA neurones with a viable, real-time fluorescent reporter. This animal model proved to be an invaluable tool to obtain the first functional study of DA cells in the olfactory bulb.
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Methods
Animals and surgical procedures
Experimental procedures were carried out so as to minimize animal suffering and the number of mice used. The procedures employed were in accordance with the Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes, and were approved by the Campus Veterinarian of the Ferrara University. A total of 72 mice have been used.
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Generation of transgenic mice (TH-GFP/21-31) was described in previous papers (Sawamoto et al. 2001; Matsushita et al. 2002). The transgene construct contained the 9.0 kb 5'-flanking region of the rat tyrosine hydroxylase (TH) gene, the second intron of the rabbit globin gene, cDNA encoding GFP, and polyadenylation signals of the rabbit globin and simian virus 40 early genes. Transgenic mice were identified by using PCR to detect tail DNA bearing the GFP sequence. Transgenic lines were maintained by breeding these animals to C57BL/6J inbred mice.
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Electrophysiological methods
Adult mice were deeply anaesthetized (I.P. injection of 60 mg kg–1 of sodium pentobarbital), decapitated, the brain was exposed, chilled with oxygenated artificial cerebrospinal fluid (ACSF), and the olfactory bulbs were dissected. Thin slices (100–150 μm) were obtained by cutting the olfactory bulb in the horizontal or in the sagittal plane, placed in the recording chamber (1 cm3 volume), and mounted on an Olympus BX50WI microscope. The slices were perfused at the rate of 2 ml min–1 with ACSF having the following composition (mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2 and 15 glucose. Saline was continuously bubbled with 95% O2–5% CO2; the osmolarity was adjusted to 305 mosmol l–1 with glucose. All drugs and neuroactive compounds were purchased from Tocris (Bristol, UK), except tetrodotoxin (Alomone, Jerusalem, Israel) and TEA (Sigma). All the substances tested were dissolved in the ACSF and perfused the entire slice preparation.
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The pipette-filling solution contained (mM): 120 KCl, 10 NaCl, 2 MgCl2, 0.5 CaCl2, 5 EGTA, 10 Hepes, 2 Na-ATP, 10 glucose; the osmolarity was adjusted at 295 mosmol l–1 with glucose and the pH was set to 7.2 with NaOH. Membrane currents were recorded and acquired with an Axopatch 200A amplifier (Axon Instruments), and a 12 bit A/D–D/A converter (Digidata 1200B; Axon Instruments); leak and capacitative transients subtraction were achieved using the P/4 protocol (Armstrong & Bezanilla, 1974); off-line analysis was performed using versions 7.0.1 and 8.0 of pCLAMP (Axon Instruments).
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The electrotonic compactness of the cells was verified by fitting a single exponential to the voltage trajectories obtained in response to hyperpolarizing pulses under current-clamp conditions. A 70–80% compensation of the series resistance was routinely used.
Cell dissociation
Adult mice (30- to 60-day-old) were used to isolate olfactory bulb neurones. Two solutions were used for the preparation: a dissecting solution and Tyrode solution. The dissecting medium (DM) contained (mM): 82 Na2SO4, 30 K2SO4, 10 Hepes, 5 MgCl2, 10 glucose, and 0.001% phenol red indicator; pH was adjusted to 7.4 with NaOH and the solution was continuously bubbled with 100% O2. Tyrode solution contained (mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5 Hepes, 20 glucose; the pH was adjusted to 7.4 with NaOH and the solution was continuously bubbled with 100% O2. Dissociation of the olfactory bulb by enzymatic digestion and mechanical trituration was performed following the procedure described by Gustincich et al. (1997), with minor changes. After dissecting and slicing the bulbs, the small pieces were transferred to a solution containing DM and 0.3% protease type XXIII (Sigma) for 30–45 min at 37°C. After enzymatic digestion, the bulbs were transferred to solution containing DM, 0.1% bovine serum albumin (Sigma) and 0.1% trypsin inhibitor (Sigma) to stop protease activity (5 min, 37°C). Bulbs were finally suspended in Tyrode solution and triturated using fire-polished Pasteur pipettes of varying gauges. The cell suspension was centrifuged at 500 g (5 min), and the pellet was resuspended in Tyrode solution. The dissociated olfactory bulb neurones were plated on a glass coverslip previously coated with concanavalin A (1 mg ml–1) to allow sedimentation of cells. The cells were allowed to set on the glass for at least 1 h before commencement of recordings. Isolated DA cells were identified under epifluorescence microscope.
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Data expressed as mean ± S.E.M. were statistically analysed using Origin 7.5 software with paired t test unless otherwise specified.
Results
Localization and general properties of TH-GFP cells
Cells expressing the GFP transgene under the TH promoter (TH-GFP) occurred primarily in the glomerular layer of the main olfactory bulb (Fig. 1A and B). The intraglomerular processes of these cells displayed high levels of TH-GFP expression, and their intertwine delimitates the glomeruli, with the soma of GFP+ cells laying around them.
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A, expression pattern of the TH-GFP transgene in the glomerular layer of the main olfactory bulb in a coronal section. GL, glomerular layer; EPL, external plexiform layer. Scale bar, 50 μm. B, magnification of the area delimitated by a dotted line in A. Scale bar, 50 μm. C, frequency distribution of the soma diameter of the cells used in this study. The distribution could be best fitted by two Gaussian curves, identifying two distinct subpopulations of cells.
Recordings with the patch-clamp technique in the whole-cell configuration were obtained from 281 DA cells in the glomerular layer. After it had been withdrawn, the pipette was always checked for the presence of fluorescence residues at the tip, as verification that the recording was actually made from a GFP+ cell.
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Cell dimensions were rather variable, as shown in Fig. 1C. Previous studies have suggested that there are two populations of DA neurones in the adult OB, based on size or location (Baker et al. 1983; Halász, 1990). In fact, the distribution of the mean cell diameter of GFP+ cells could be best fitted with two Gaussian curves, identifying two subpopulations with average sizes of 5.67 ± 0.96 and 9.48 ± 2.39 μm (R2 = 0.991); the same result could be obtained from the analysis of the membrane capacitances, whose frequency distribution could be best fitted by two Gaussians (5.41 ± 1.5 and 10.63 ± 3.45 pF, R2 = 0.975, not shown). However, we found no significant differences in the properties of the two populations.
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About 80% of DA neurones were spontaneously active. In the cell-attached configuration, action currents were recorded across the patch, usually structured in a regular, rhythmic pattern (Fig. 2A) with an average frequency of 7.30 ± 1.35 Hz (n = 31). After disruption, about 60% of the cells continued to fire spontaneous action potentials under current-clamp conditions (Fig. 2B) without any significant alteration of the firing frequency (7.84 ± 2.44 Hz, n = 14). Interspike intervals were rather constant in most of the cells (Fig. 2C), and irregular in others for the presence of sporadic misses. Occasionally, especially in isolated cells (see below), the firing was structured in bursts. We found no correlation of the firing frequency with cell size.
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A, action currents in cell-attached mode. B, action potentials in whole-cell mode. C, frequency distribution of the inter-event time for the cell shown in B. D, frequency of spontaneous firing in TH-GFP cells under the indicated experimental conditions. CA, cell attached; WC, whole cell.
This spontaneous activity was completely blocked by TTX (0.3 μM) or by Cd2+ (100 μM), but persisted after block of glutamatergic and GABAergic synaptic transmission with kynurenate (1 mM) and picrotoxin (50 μM), suggesting that it was due to intrinsic properties of the cell membrane and was not driven by external synaptic inputs.
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Occasionally we did observe spontaneous synaptic currents, which were completely blocked by a mixture of 1 mM kynurenate and 50 μM picrotoxin (not shown), and which were not further investigated for the purpose of this study.
We studied the dopaminergic neurones under current- and voltage-clamp conditions to characterize the ionic currents underlying spontaneous firing. In voltage-clamped neurones, currents were elicited both by step and ramp depolarizations.
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Depolarization activates a complicated pattern of current flow, in which a variety of conductances coexist, the most prominent of which were a fast transient sodium current and a non-inactivating potassium current (Fig. 3A and B). We identified specific ionic currents present in the cells by measurements of their voltage dependence and kinetics during step depolarizations, together with ionic substitution and blocking agents to isolate individual components of the currents. After block of the potassium currents, obtained by adding 20 mM TEA in the perfusing solution and by equimolar substitution of internal K+ ions with Cs+, a persistent inward current was observed after the fast transient inward current had completely subsided (Fig. 3C and D). The amplitude of this persistent component, measured as the average of the current amplitude during the last 10 ms of the depolarizing step, had a maximum amplitude of 223.3 ± 32.2 pA (n = 21) at –20 mV, and could be separated into two components, sustained by sodium and calcium ions (see below).
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A and B, voltage-clamp recordings from the same cell, in normal saline, held at –70 mV (A) and at –50 mV (B), and depolarized to potentials ranging from –50 to +50 mV. C, inward currents recorded under voltage-clamp conditions in response to depolarizing steps ranging from –80 to +50 mV; holding potential was –100 mV. Potassium currents were suppressed by ionic substitution of intracellular K+ ions with Cs+, and addition of 20 mM TEA in the extracellular medium. The inset shows the current–voltage relationship of the persistent inward current, averaged at the times indicated by the box. D, details of some of the traces shown in C, at higher magnification, to show the persistent inward current.
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Spontaneous activity
The presence of autorhythmic activity was the most salient feature of DA cells in the olfactory bulb, so the first efforts were aimed at elucidating the underlying mechanisms. We first tried to understand if the spontaneous activity was due to the presence of pacemaker currents or to synaptic mechanisms reverberating excitation from one cell to the other. Dissociated TH-GFP cells conserved their capacity of generating rhythmic activity, clearly indicating that this is an intrinsic property of these cells. Dissociated TH-GFP cells showed a spontaneous frequency of firing of 13.57 ± 1.79 (n = 24) and 15.75 ± 3.12 H2 (n = 14) in whole-cell and cell-attached modes, respectively (Fig. 2D). This frequency was about double the corresponding value observed in thin slices, suggesting the existence in semi-intact tissue of some inhibitory control, possibly autoinhibition, which has not been further investigated in this study.
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A likely candidate for the pacemaking process was the inward rectifier current (Ih), which we have shown to be present in a subpopulation of PG cells in a previous report (Cadetti & Belluzzi, 2001). However, we failed to observe any trace of a hyperpolarization-activated current in TH-GFP cell in the olfactory bulb (not shown), so we focused on non-inactivating inward currents.
Persistent sodium current
In DA cells, after the fast transient Na+ current had completely subsided, a persistent inward current showing no sign of inactivation after 200 ms was observed.
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We first applied TTX (0.3–1.2 μM), which suppressed a significant fraction of the persistent inward current, indicating it received a contribution from a non-inactivating, TTX-sensitive channel. This current–voltage relationship was virtually coincident with the residual persistent current measured after treatment with 100 μM Cd2+ (see below), and therefore the data were pooled. The current–voltage relationship of the fraction of current abolished by TTX or remaining after Cd2+ treatment is shown in Fig. 4A. The persistent sodium current, INa(P), was activated at potentials more negative than –70 mV, and reached a maximum amplitude of –27.5 ± 2.97 pA at –30 mV. The corresponding conductance–voltage relationship, calculated by dividing the current amplitude by the sodium driving force, could be fitted by a Boltzmann equation with a midpoint at –48.8 mV and a slope of 6.51 mV nS–1 (Fig. 4B). The maximum value of gNa(P) conductance was 0.41 nS, about 200 times smaller than that of the fast sodium current (INa(F), see below), but contrary to the latter, this current is activated in the pacemaker range, showing an amplitude of –7.3 pA at –60 mV.
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A, I–V relationship. Pooled data obtained as fraction of non-inactivating current suppressed by TTX (n = 12) and residual persistent current after Cd2+ block (n = 6). B, conductance–voltage relationship of persistent sodium current, obtained from the mean data shown in A. The continuous curve is the Boltzmann fit, with upper asymptote of 0.41 nS, midpoint at –48.8 mV and slope of 6.51 mV nS–1.
The calcium currents
After block of the TTX-sensitive component and suppression of the K+ current by equimolar substitution of intracellular K+ with Cs+, a persistent inward current could be observed at the end of prolonged depolarizing steps (Fig. 5A, upper traces). This residual fraction could be almost completely blocked by Cd2+ or Co2+ ions (Fig. 5A, lower traces), suggesting that this second component was sustained by calcium ions. The very small fraction of current remaining after TTX and Cd2+ block has not been further investigated in the present study.
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A, calcium current recorded in response to depolarizing voltage steps to potentials ranging from –70 to +10 mV from a holding potential of –100 mV. Above: tracings recorded in the presence of 1.2 μM TTX, 20 mM TEA in the extracellular solution, and with Cs+ as a substitute for K+ ions in the intracellular solution. Lower tracings were recorded under the same conditions after addition of 100 μM Cd2+. B, barium currents recorded in the same conditions described for A. C, I–V relationship of Ca2+ and Ba2+ currents from a 10 neurone sample from thin slices. D, activation time constant, measured from a 10 neurone sample by fitting a single exponential to the rising phase of the current. The continuous line is described by the equation: aCa(L) = 1.23 + exp(–V/74.6). E, effect of 10 μM nifedipine on calcium current in a group of six TH-GFP PG cells in slices. F, histogram showing the effect of nifedipine (10 μM, cells shown in E) and calcicludine (1 μM, not shown, n = 7) measured at –10 mV.
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Using classical pharmacological tools, ionic substitutions and voltage-clamp protocols, we could dissect the voltage-dependent Ca2+ currents, Cav, into several components, including both high-voltage activated (HVA) and low-voltage activated (LVA) currents.
The larger of these components, by its overall kinetics, its voltage dependence and the absence of inactivation, was identified as a possible L-type Ca+ current. Its properties were studied in slices, after blockage of the Na+ currents with 0.3–1.2 μM TTX and of the K+ currents by equimolar substitution with Cs+ in the pipette-filling solution and 20 mM TEA in the perfusing bath. The protocols used were either voltage steps or voltage ramps, giving virtually identical results. The I–V relationship of the Ca+ current (Fig. 5C), measured in a 10 neurone sample averaging the last 5 ms at the end of a 40 ms depolarizing step, had a maximum amplitude of –108.7 ± 11.9 pA at –10 mV. The corresponding conductance–voltage relationship showed a maximum conductance of 2.3 nS, with a midpoint at –25.6 mV.
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Equimolar substitution of Ca2+ with Ba2+ increased the amplitude of this current by a factor of about 3 (Fig. 5C), without changing the time constant of activation. On this current we tested the effects of two blockers of L-type calcium channels, nifedipine and calcicludine. The fraction of current blocked by the two drugs at different voltages was quantified by subtraction of I–V data acquired before and after treatment.
The effects of 10 μM nifedipine on peak Ca2+ current amplitude was assessed in six PG cells (Fig. 5E and F). On average, the drug blocked 61.1 ± 14% of the current measured at the point of its maximum amplitude (–10 mV).
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A 60 amino acid peptide, calcicludine (CaC), has been described as having a powerful effect on all type of high-voltage-activated Ca2+ channels (L-, N- and P-type) (Schweitz et al. 1994). Since one of the regions of the CNS presenting the highest densities of 125I-labelled CaC binding sites is the glomerular layer of the olfactory bulb (Schweitz et al. 1994), we tested the ability of this toxin in suppressing the non-inactivating Ca2+ current found in the DA neurones. CaC (1 μM) was much more effective than nifedipine, with an inhibitory action averaging 72.7 ± 3.13%.
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We also tried to define whether other types of HVA neuronal Cav channels (P/Q-, N-) were present in TH-GFP cells. Using classical blockers, like -conotoxin GVIA (0.82 μM) that blocks the N-type, or spider toxin -agatoxin IVA (10 nM) that blocks P/Q-type channels, we observed the suppression of a fraction of HVA Ca2+ current remaining after nifedipine block (at –10 mV 38 and 42%, respectively, not shown), suggesting the presence of limited amounts of the corresponding HVA Ca2+ channels.
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Since the long-lasting HVA Cav currents were not directly involved in the pacemaker process (see below), and for the dominance of L- over N- and P/Q-type current, for the purpose of the numerical reconstruction of the electrical activity of these cells (see below), they were kinetically modelled as a unique, non-inactivating component. The rising phase of the current was fitted by a single exponential, with a time constant of 1.2 ± 0.3 ms at 0 mV (n = 10, see legend of Fig. 5D for further details).
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Since in situ hybridization experiments have localized the expression of two transcripts (1G and 1I) of the T-type calcium channel in the glomerular layer of the olfactory bulb (Talley et al. 1999; Klugbauer et al. 1999), we checked for the presence of LVA Ca2+ current. Unfortunately several characteristics of these channels hampered their study in our preparation. First, contrary to the cardiac cells or transfected cells, in TH-GFP interneurones this current is small: we have calculated a maximum conductance of 0.35 nS, corresponding to a peak current of about 20 pA at –35 mV. The problem was further complicated by the fact that on one hand it was difficult to get accurate space clamping in slices, and on the other hand, in isolated cell preparations the current was difficult to resolve, probably because of the preferential localization of these channels on the dendrites (Perez-Reyes, 2003). Second, the conductance of these channels cannot be significantly increased by substitution of Ca2+ with Ba2+ ions, as it can be done with HVA channels. Third, there are no effective pharmacological tools for the study of T-type Ca2+ channels, because they are relatively resistant to most organic calcium channel blockers, such as dihydropyridines, that block the L-type, or peptide toxins, such as -conotoxin or -agatoxin.
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Despite these difficulties, we succeeded in isolating a T-type calcium current in dissociated cells (Fig. 6). The protocol used was a rapid ramp (7 V s–1) from –100 to 40 mV, in the presence of TTX (1 μM) and after substitution of Ca2+ with Sr2+, which is known to have a slightly higher permeability than Ca2+ in T-type Ca2+ channels (Takahashi et al. 1991). Under these conditions, an inward inflection peaking at about –40 mV, distinct from the peak due to the L-type Ca2+ channels (Fig. 6A), could be seen. Nickel is a non-selective inhibitor of calcium channels, but transient low voltage-activated (LVA) T-type Ca2+ channels are particularly sensitive (IC50 < 50 μM) (Perchenet et al. 2000; Wolfart & Roeper, 2002), whereas other HVA Cav channels (L-, P/Q and N-type) are less sensitive (IC50 > 90 μM) (Zhang et al. 1993; Randall, 1998). In fact 100 μM nickel did selectively eliminate the first peak, leaving the second unaltered. The difference, averaged in three cells, is shown in Fig. 6B, and the conductance, calculated assuming a Ca2+ equilibrium potential of 45 mV, is illustrated in Fig. 6C. The current activates at potentials positive to –65 mV and peaks at about –35 mV, with a maximum conductance of 0.35 nS. The point of half-activation is –45.3 mV, which is in line with the known values for this current (Perez-Reyes, 2003).
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A, voltage-clamp ramps were performed from –80 to +40 mV at a speed of 7 V s–1 in the presence of TTX (1 μM), and after substitution of Sr2+ for Ca2+; the traces were corrected for leakage. Under these conditions, a distinct bump can be seen at –40 mV, preceding the HVA calcium current (here peaking at –10 mV), which is selectively suppressed by 100 μM Ni2+. B, transient Ca2+ current generated using the protocol described in A calculated by subtracting the current–voltage curves in control and in the presence of 100 μM Ni2+. The low-Ni2+ sensitive current (average from three cells) is activated at membrane potentials more positive than –60 mV. C, conductance–voltage relationship of the low-Ni2+ sensitive current. The continuous line is a Boltzmann fit, with a midpoint at –45.3 mV and a maximum conductance of 0.35 nS. All recordings in this figure were performed from spontaneously bursting dissociated TH-GFP cells.
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The pacemaker currents
We next tried to elucidate the ionic basis of the pacemaker current underlying the spontaneous firing. We first found that the Ca2+ current is involved in the pacemaker process, as 100 μM Cd2+ completely and reversibly blocked the spontaneous firing (Fig. S1A and B in Supplemental material). Then, using a panel of different Cav channels inhibitors, we tried to define which types of neuronal Cav channels (L-, P/Q-, N-, R- and T-type) contributed to the pacemaker current.
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The classical selective L-type Ca2+ channel antagonist nifedipine (10 μM), which blocked the long-lasting Ca2+ current by about two thirds (Fig. 5E and F), had no effect at all in the spontaneous firing frequency, either in cell-attached mode (Fig. S1C in Supplemental material) or in whole-cell configuration, in a total of six cells recorded in slices. Also calcicludine (1 μM), a powerful although less selective HVA channel blocker (Schweitz et al. 1994), which inhibited the long-lasting Ca2+ channel component (73%, Fig. 5F), was equally ineffective, even after very long periods of application (Fig. S1D and E in Supplemental material). Analogous results were obtained using other classical blockers of HVA Ca2+ channels, like -conotoxin GVIA (0.82 μM), which blocks the N-type, or spider toxin -agatoxin IVA (10 nM) which blocks P/Q-type channels. Both did suppress a fraction of the residual HVA Ca2+ current after nifedipine block (at –10 mV 38 and 42%, respectively), suggesting the presence of the corresponding types of HVA Ca2+ channels, but none of them affected the spontaneous firing (not shown).
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Among the remaining candidates for a role in pacemaking was the LVA, T-type channel. Mibefradil has been reported to inhibit T-type calcium channel current in several neuronal types (Randall & Tsien, 1997; Viana et al. 1997; Todorovic & Lingle, 1998). We therefore used this drug, which proved to be considerably powerful in blocking the spontaneous activity of PG DA neurones, both in cell attached and in whole-cell mode (Fig. S2A and B in Supplemental material). Mibefradil (5–10 μM) completely and reversibly blocks the spontaneous activity, inducing an evident hyperpolarization that on average amounted to about 15 mV (Fig. S2B). Nickel (100 μM), that we have shown to be a selective blocker of T-type calcium current in these cells (Fig. 6A), induced a reversible block of spontaneous firing, also accompanied by a hyperpolarization of 10–15 mV (Fig. S2C), an effect almost superimposable on that observed with mibefradil, further confirming a role of ICa(T) in the pacemaking process.
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Isolation and kinetic characterization of the fast transient Na+ current
A classical fast transient, TTX-sensitive sodium current was present in all the DA neurones studied. The current was isolated by blocking the Ca2+ current with 100 μM Cd2+, and by equimolar substitution of intracellular K+ with Cs+; in addition, the K+ channels were blocked by adding 20 mM TEA in the perfusing solution (and occasionally also in the intracellular solution to complete the blockade). On this current we performed a standard Hodgkin-Huxley-type analysis (Hodgkin & Huxley, 1952), and the relevant results are summarized in the Appendix. Representative recordings, with an explanation of the protocols used, are shown in the Supplemental material (Fig. S3, and relative legend).
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Isolation and time course of the potassium currents
A delayed rectifier-type potassium current was present in TH-GFP cells (e.g. Figure 3B) which has been kinetically characterized. The current, isolated by blocking the sodium current with TTX, was calcium dependent only for a small part (at 0 mV the fraction suppressed by Cd2+ 100 μM was about 10%), and thus it has been modelled as a single component. The equations describing the time- and voltage-dependence of the potassium current are given in the Appendix and illustrated in Fig. S4 (in Supplemental material).
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Modelling the natural burst firing in bulbar DA neurones
Finally, we have modelled the bulbar DA neurones in Hodgkin-Huxley terms (Hodgkin & Huxley, 1952), considering the cell as a single electrical and spatial compartment. As for the conductances considered, we incorporated the two sodium currents (fast transient and persistent), the L-type Ca2+ current, the delayed rectifier K+ current, all according to the experimental data presented above, and the T-type calcium current. Since our characterization of this current was incomplete (and because of the difficulty in obtaining a complete kinetic description of this current), in our model we have integrated our data on the activation process with others, concerning the inactivation, derived from the literature (Wang et al. 1996; Perez-Reyes, 2003), and consistent with our experimental data.
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All the equations and parameters used, as well as the assumptions made, are listed in the Appendix. The solution of the set of differential equations describing the kinetics of the currents considered lead to the tracings reported in Fig. 7. The model we set up shows intrinsic spiking capabilities with full-size action potentials at the same frequencies observed in TH-GFP cells.
A, voltage tracings. B, current tracings including the five conductances: INa(F), INa(P), ICa(T), ICa(L), IK(V). C and D, enlargement of the last two events of A and B to show the pacemaking process. In D the outward current has been omitted, and the inward currents have been amplified by a factor of about 500 with respect to B.
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The model is crucial to understand the interplay of the currents underlying the pacemaking process. As it can be clearly seen in Fig. 7C, during the interspike interval, the currents which cause the progressive depolarization of the cell are, in order, the T-type calcium current, and then the persistent sodium current. Although these currents are amazingly small in amplitude (max. 4 pA) compared with fast transient sodium and delayed rectifier potassium currents associated to the action potential (about 1 nA), they are nevertheless sufficient to depolarize these cells, due to their rather high input resistance (about 700 M).
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It is the T-type calcium current which sets in motion the depolarizing process but, both INa(P) and ICa(T) are necessary to sustain spontaneous firing as the selective block of one or both abolish spontaneous activity: the model cell is still capable of responding with a single action potential to the injection of a depolarizing current pulse, but it fails to fire repetitively. The T-type calcium conductance amplitude is critical in determining the firing frequency: small changes in its value (from 0.35 to 0.4 nS) are sufficient to drive the intrinsic spiking from 8 to 16 Hz.
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The model thus confirms that, in addition to INa(P), another component is necessary to sustain repetitive firing: a T-type calcium current. Together with the experimental finding that treatments which block ICa(T), such as mibefradil and nickel at micromolar concentrations, are both capable of preventing repetitive firing, it therefore appears that this current is an essential component of the pacemaking process.
Discussion
This study represents the first description of the functional properties of DA neurones in the mammalian olfactory bulb. The animal model used for these experiments, a strain of transgenic mice expressing a reporter protein under the TH promoter (Sawamoto et al. 2001; Matsushita et al. 2002), allows an easy identification of DA neurones both in thin slices and dissociated cells, proving to be a superb tool for targeting live DA neurones in electrophysiological studies. The main results obtained are the demonstration that DA neurones in the OB are autorhythmic, and the description of the interplay of subthreshold currents underlying intrinsic spiking.
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Distribution and general properties of DA neurones
In mammalians, neurones expressing high levels of the reporter protein were found in the glomerular layer, as expected from abundant literature, indicating that this is the only region of the main olfactory bulb where TH is expressed (Halász, 1990; Kratskin & Belluzzi, 2003). A previous study in the same mice strain has demonstrated an overlapping expression of the fluorescent reporter and TH protein in olfactory bulb only in those DA neurones that received afferent stimulation from receptor cells (Baker et al. 2003).
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DA neurones in the mice OB have a complement of voltage-dependent currents, which have been kinetically characterized in this study. Among these, the persistent Na+ current deserves some comment. Many neurones in the mammalian CNS have a non-inactivating component of the TTX-sensitive sodium current (Crill, 1996). Although its magnitude in bulbar DA neurones is about 0.5% of the transient sodium current, INa(P) appears to have an important functional significance because it is activated at potentials 8–10 mV more negative than the transient sodium current. At this potential few voltage-gated channels are activated and the neurone input resistance is high. The conductance–voltage relationship for gNa(P) in TH-GFP DA neurones has a half-activation point at –47.7 mV, very close to the values found in hippocampal CA1 neurones (French et al. 1990) and pyramidal neurones of the neocortex (Brown et al. 1994). Although this current might appear small, 7.3 pA at –60 mV (Fig. 4A), it suffices to depolarize the cell membrane of these cells, which have an average input resistance of 700 M, by about 5 mV.
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We have considered the possibility that INa(P) is in fact a window current, the steady current predicted by the HH model and arising from overlap of the steady-state activation and inactivation curves for sodium conductance (Attwell et al. 1979; Colatsky, 1982). Based on the kinetic analysis of the fast Na+ current (Supplemental material, Fig. S3H), we calculated the window current at different potentials. The numerical simulation indicates this current does provide a measurable contribution to the TTX-sensitive persistent inward current. However, this contribution, which is maximal at –45 mV, falls to virtually zero at –30 mV, the potential at which the persistent sodium current displays its maximum amplitude (Fig. 4A). In other words, INa(P) and the window current of INa(F) develop at different potentials, and therefore are distinct currents.
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Interaction of ionic currents to produce spontaneous firing
The pharmacological treatments, ion substitution experiments, kinetic analysis and numerical simulations, allow a rather precise understanding of mechanisms underlying spontaneous firing in TH-GFP cells. The first observation is that the slow depolarization between spikes is sustained by the persistent, tetrodotoxin-sensitive sodium current and by the T-type calcium current, without any intervention of hyperpolarization-activated, h-type current. This is of some interest, as the h-current has been described in cells of the glomerular layer (Cadetti & Belluzzi, 2001). However, if it is true that in some dopaminergic midbrain neurones the h-channels have been shown to be actively involved in pacemaker frequency control (Seutin et al. 2001; Neuhoff et al. 2002), the same has not been observed in many other dopaminergic neurones, as in the retina (Feigenspan et al. 1998), or in the ventral tegmental area (Neuhoff et al. 2002).
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The role of a calcium current in rhythm generation is revealed by the rapid and reversible block of the spontaneous firing by Cd2+, both in slices and in dissociated cells (Supplemental material, Fig. S1B). However, among the many HVA Ca2+ channels present in the DA neurones of the olfactory bulb (a large L-, and smaller N- and P/Q types), none proved to be effective in the control of spontaneous firing. On the contrary, conditions which are known to block the LVA T-type Ca2+ channels (mibefradil and nickel in the micromolar range) did break off the spontaneous activity.
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To better understand the interplay of conductances underlying the spontaneous activity, we developed a numerical HH-type model (Hodgkin & Huxley, 1952) of TH-GFP cells, based on the kinetic characterization of the voltage-dependent currents described above, which reproduces their behaviour fairly well. This model is useful not only for the validation of measurements and analysis, but, first and foremost, because it provides an in-depth quantitative description of the events underlying the pacemaking process (Fig. 7C).
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Both INa(P) and ICa(T) are necessary to sustain spontaneous firing as the selective block of one or both abolish spontaneous activity. However, it is the T-type calcium current which sets in motion the depolarizing process: although essential to the rhythm generation, INa(P) replaces ICa(T) only in the second half of the depolarizing phase.
Small changes in the parameters of INa(F) and IK(V), such as the half-activation point shift of a few millivolts, were sufficient to arrest spontaneous firing but did not affect the capacity to respond with single action potentials to depolarizing stimuli. This proves that the pacemaking process is due to an interplay of conductances which are in a delicate and precise equilibrium and, furthermore, it confirms that the model is capable of capturing the essential features of the excitability profile of these cells.
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In conclusion, the experimental observation that the block of ICa(T) with mibefradil or nickel at micromolar concentrations are both capable of preventing repetitive firing are well explained by the model, which assigns to this current the role of primum movens in the pacemaking process.
Significance to olfactory function
Our results, and especially the indication that DA neurones in the glomerular layer are autorhythmic, open the field to many speculations.
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As indicated by an abundant literature, dopamine is inhibitory in the olfactory bulb, acting primarily on presynaptic D2 receptors located in the olfactory nerve terminal (Brünig et al. 1999; Hsia et al. 1999; Wachowiak & Cohen, 1999; Berkowicz & Trombley, 2000; Ennis et al. 2001; Davila et al. 2003). This inhibitory role has been observed both in mammalians and in the frog olfactory bulb, although in the latter both anatomical localization of D2-like receptors and functional data on dopamine involvement in information processing differ in several aspects from those reported in mammals (Duchamp-Viret et al. 1997; Davison et al. 2004).
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In mammalian systems, it is known that the glomerular neuropile, far from being a homogeneous structure, shows a complex subcompartimental organization: within a single glomerulus, olfactory nerve (ON) islets delimit areas in which dendritic branches receive sensory input from ON terminals, and are well separated from non-ON zones, from which ON terminals are excluded (Chao et al. 1997; Kasowski et al. 1999; Toida et al. 2000). Some TH-immunoreactive PG cells arborize on the ON islets, others in the non-ON zone (Kosaka et al. 1997). Although it has been reported that in some mouse strains TH-positive cells are not in contact with ON terminals (Weruaga et al. 2000), in the strain used for these experiments – one of the most common (C57BL/6 J) – such contacts have been demonstrated (T. Kosaka and K. Kosaka, personal communication).
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The glomerular compartmentalization supports the hypothesis that information processing is subdivided regionally within the mammalian glomerulus (Kasowski et al. 1999). DA neurones establishing contacts in ON- or in non-ON zones, possibly play different roles. Within the ON zones, dendrites of DA neurones receive excitatory synapses from ON axon terminals (Chao et al. 1997; Kasowski et al. 1999; Toida et al. 2000). It has been reported that D2 dopaminergic receptors are located in ON terminals (Levey et al. 1993; Coronas et al. 1997), and electrophysiological studies have shown that their activation can reduce the probability of glutamate release, and hence the excitation of projection neurones (Duchamp-Viret et al. 1997; Hsia et al. 1999; Berkowicz & Trombley, 2000; Ennis et al. 2001; Davison et al. 2004). In fact, the most significant impact of DA neurones is expected at the level of the synaptic triad formed by the ON and the dendrites of mitral/tufted (MT) cells and PG cells, where DA neurones directly control the input of projection neurones from receptor cell axons (Brünig et al. 1999). Since the ON islets appear to be further segregated from the rest of the glomerular neuropile due to the presence of ‘glial wraps’ (Kasowski et al. 1999), the spontaneous activity of DA neurones (which implies continuous release of dopamine within a restricted space), would create a condition of tonic inhibition of the ON. The situation in the frog olfactory bulb appears entirely different, in that DA neurones directly inhibit the projection neurones (Duchamp-Viret et al. 1997; Davison et al. 2004).
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In addition, DA neurones send their dendrites also into non-ON zones, where they contact dendrites of projection neurones and interneurones, and centrifugal axons. Projection neurones express D1 and D2 receptors (Brünig et al. 1999; Davila et al. 2003), and it has been shown that dopamine exerts a complex modulatory action between them and interneurones (ibidem), an effect that also in this case would be amplified by the restricted space of the glomerular neuropile. Within this framework, dopamine might play a central role in the processing of olfactory information by acting at two levels: it would control the input of the sensory signal, and it would modulate the mechanism of GABAergic inhibition (Brünig et al. 1999; Davila et al. 2003).
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It remains to be explained why DA neurones in the glomerular region of the OB are among the very few in the mammalian CNS which are generated also in adulthood. This raises a series of questions concerning the mechanisms controlling their migration and differentiation, and, also, it opens interesting perspectives for the exploitation of the olfactory bulb as a source of undifferentiated DA cells that could be expanded ex vivo and used for transplants in neurodegenerative diseases.
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Supplemental material
The online version of this paper can be accessed at:DOI: 10.1113/jphysiol.2005.084632
http://jp.physoc.org/cgi/content/full/jphysiol.2005.084632/DC1
and contains supplemental figures.
This material can also be found at:http://www.blackwellpublishing.com/products/journals/suppmat/tjp/tjp821/tjp821/sm.htm
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Cable properties
Input resistance: 706 M; membrane capacitance 6.7 pF
Whole-cell conductances (nS)
Equilibrium potentials (mV)
Fast transient sodium current – INa(F)
Steady-state activation:
Steady-state inactivation:
, http://www.100md.com Activation time constant:
Inactivation time constant:
De-inactivation time constant: 42 ms
Delayed rectifier potassium current – IK(V)
Steady-state activation:
Activation time constant:
Deactivation time constant:
T-type calcium current – ICa(T)
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Steady-state activation:
Steady-state inactivation:
Activation time constant:
Inactivation time constant:
Persistent sodium current – INa(P)
Steady-state activation:
Activation time constant: 0.9 ms
L-type calcium current – ICa(L)
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Steady-state activation:
Activation time constant:
Deactivation time constant: 0.587 ms
The reconstruction has been made using time increments of 83.3 μs, corresponding to a frequency of 12 kHz.
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2 Fukushima Medical University School of Medicine, Department Molecular Genetics, Hikarigaoka, Fukushima 960-1295, Japan
3 Keio University, School of Medicine, Department of Physiology, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Abstract
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The olfactory bulb of mammals contains a large population of dopaminergic interneurones within the glomerular layer. Dopamine has been shown both in vivo and in vitro to modulate several aspects of olfactory information processing, but the functional properties of dopaminergic neurones have never been described due to the inability to recognize these cells in living preparations. To overcome this difficulty, we used a transgenic mouse strain harbouring an eGFP (enhanced green fluorescent protein) reporter construct under the promoter of tyrosine hydroxylase, the rate-limiting enzyme for cathecolamine synthesis. As a result, we were able to identify dopaminergic neurones (TH-GFP cells) in living preparations and, for the first time, we could study the functional properties of such neurones in the olfactory bulb, in both slices and dissociated cells. The most prominent feature of these cells was the autorhythmicity. In these cells we identified five main voltage-dependent conductances: the two having largest amplitude were a fast transient Na+ current and a delayed rectifier K+ current. In addition, we observed three smaller inward currents, sustained by Na+ ions (persistent type) and by Ca2+ ions (LVA and HVA). Using pharmacological tools and ion substitution methods we showed that the pacemaking process is supported by the interplay of the persistent Na+ current and of a T-type Ca2+ current. We carried out a complete kinetical analysis of the five conductances present in these cells, and developed a Hodgkin-Huxley model of TH-GFP cells, capable of reproducing accurately the properties of living cells, including autorhytmicity, and allowing a precise understanding of the process.
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Introduction
In the mammalian olfactory bulb (OB) dopaminergic (DA) neurones constitute a fraction of the cells occupying the most external (glomerular) layer (Halász et al. 1977). In this region, populated by three types of interneurones, periglomerular (PG) cells, short-axon cells and external tufted (ET) cells (Halász, 1990) – often collectively referred to as juxtaglomerular cells – an estimated 10% of the neurones in adulthood are positive for tyrosine hydroxylase (TH) (McLean & Shipley, 1988; Kratskin & Belluzzi, 2003), the rate-limiting enzyme for dopamine synthesis. Dopaminergic neurones in the glomerular layer include PG cells (Kosaka et al. 1985; Gall et al. 1987) and a fraction of ET cells (Halász, 1990). Several studies have focused on the role of dopamine in the olfactory bulb, using immunohistochemical (Baker et al. 1983; Guthrie et al. 1991), behavioural (Doty & Risser, 1989) and electrophysiological techniques (Nowycky et al. 1983; Ennis et al. 2001; Davila et al. 2003). Despite the wealth of information available on the role of dopamine in olfaction, the functional properties of DA neurones in the OB remains entirely unknown.
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A property shared by many DA neurones in the CNS is their capacity to generate rhythmic action potentials even in the absence of synaptic inputs (Grace & Onn, 1989; Hainsworth et al. 1991; Yung et al. 1991; Feigenspan et al. 1998; Neuhoff et al. 2002). In this paper we show for the first time that DA cells in the glomerular layer of the olfactory bulb possess a pacemaker activity, and we provide an explanation for the ionic basis of rhythm generation in these cells.
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There is an additional reason to study the functional properties of DA neurones in the OB other than their role in olfaction. The olfactory bulb is one of the rare regions of the mammalian CNS in which new cells, derived from stem cells in the anterior subventricular zone, are also added in adulthood (Gross, 2000). In the OB, these cells differentiate in interneurones in the granular and glomerular layers. Among these cells there are DA neurones (Betarbet et al. 1996; Baker et al. 2001), and this has raised a remarkable interest because, with their accessibility, they could provide a convenient source of autologous DA neurones for transplant therapies in neurodegenerative diseases, like Parkinson's disease.
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Electrical recordings from identified DA neurones have been largely impeded by the difficulty of discerning these cells in living brain tissue. Our experimental approach to specifically target DA neurones for electrophysiological analysis was to use transgenic mice carrying green fluorescent protein (GFP) under the control of TH promoter (TH-GFP cells) (Sawamoto et al. 2001; Matsushita et al. 2002), thereby tagging live DA neurones with a viable, real-time fluorescent reporter. This animal model proved to be an invaluable tool to obtain the first functional study of DA cells in the olfactory bulb.
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Methods
Animals and surgical procedures
Experimental procedures were carried out so as to minimize animal suffering and the number of mice used. The procedures employed were in accordance with the Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes, and were approved by the Campus Veterinarian of the Ferrara University. A total of 72 mice have been used.
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Generation of transgenic mice (TH-GFP/21-31) was described in previous papers (Sawamoto et al. 2001; Matsushita et al. 2002). The transgene construct contained the 9.0 kb 5'-flanking region of the rat tyrosine hydroxylase (TH) gene, the second intron of the rabbit globin gene, cDNA encoding GFP, and polyadenylation signals of the rabbit globin and simian virus 40 early genes. Transgenic mice were identified by using PCR to detect tail DNA bearing the GFP sequence. Transgenic lines were maintained by breeding these animals to C57BL/6J inbred mice.
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Electrophysiological methods
Adult mice were deeply anaesthetized (I.P. injection of 60 mg kg–1 of sodium pentobarbital), decapitated, the brain was exposed, chilled with oxygenated artificial cerebrospinal fluid (ACSF), and the olfactory bulbs were dissected. Thin slices (100–150 μm) were obtained by cutting the olfactory bulb in the horizontal or in the sagittal plane, placed in the recording chamber (1 cm3 volume), and mounted on an Olympus BX50WI microscope. The slices were perfused at the rate of 2 ml min–1 with ACSF having the following composition (mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2 and 15 glucose. Saline was continuously bubbled with 95% O2–5% CO2; the osmolarity was adjusted to 305 mosmol l–1 with glucose. All drugs and neuroactive compounds were purchased from Tocris (Bristol, UK), except tetrodotoxin (Alomone, Jerusalem, Israel) and TEA (Sigma). All the substances tested were dissolved in the ACSF and perfused the entire slice preparation.
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The pipette-filling solution contained (mM): 120 KCl, 10 NaCl, 2 MgCl2, 0.5 CaCl2, 5 EGTA, 10 Hepes, 2 Na-ATP, 10 glucose; the osmolarity was adjusted at 295 mosmol l–1 with glucose and the pH was set to 7.2 with NaOH. Membrane currents were recorded and acquired with an Axopatch 200A amplifier (Axon Instruments), and a 12 bit A/D–D/A converter (Digidata 1200B; Axon Instruments); leak and capacitative transients subtraction were achieved using the P/4 protocol (Armstrong & Bezanilla, 1974); off-line analysis was performed using versions 7.0.1 and 8.0 of pCLAMP (Axon Instruments).
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The electrotonic compactness of the cells was verified by fitting a single exponential to the voltage trajectories obtained in response to hyperpolarizing pulses under current-clamp conditions. A 70–80% compensation of the series resistance was routinely used.
Cell dissociation
Adult mice (30- to 60-day-old) were used to isolate olfactory bulb neurones. Two solutions were used for the preparation: a dissecting solution and Tyrode solution. The dissecting medium (DM) contained (mM): 82 Na2SO4, 30 K2SO4, 10 Hepes, 5 MgCl2, 10 glucose, and 0.001% phenol red indicator; pH was adjusted to 7.4 with NaOH and the solution was continuously bubbled with 100% O2. Tyrode solution contained (mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5 Hepes, 20 glucose; the pH was adjusted to 7.4 with NaOH and the solution was continuously bubbled with 100% O2. Dissociation of the olfactory bulb by enzymatic digestion and mechanical trituration was performed following the procedure described by Gustincich et al. (1997), with minor changes. After dissecting and slicing the bulbs, the small pieces were transferred to a solution containing DM and 0.3% protease type XXIII (Sigma) for 30–45 min at 37°C. After enzymatic digestion, the bulbs were transferred to solution containing DM, 0.1% bovine serum albumin (Sigma) and 0.1% trypsin inhibitor (Sigma) to stop protease activity (5 min, 37°C). Bulbs were finally suspended in Tyrode solution and triturated using fire-polished Pasteur pipettes of varying gauges. The cell suspension was centrifuged at 500 g (5 min), and the pellet was resuspended in Tyrode solution. The dissociated olfactory bulb neurones were plated on a glass coverslip previously coated with concanavalin A (1 mg ml–1) to allow sedimentation of cells. The cells were allowed to set on the glass for at least 1 h before commencement of recordings. Isolated DA cells were identified under epifluorescence microscope.
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Data expressed as mean ± S.E.M. were statistically analysed using Origin 7.5 software with paired t test unless otherwise specified.
Results
Localization and general properties of TH-GFP cells
Cells expressing the GFP transgene under the TH promoter (TH-GFP) occurred primarily in the glomerular layer of the main olfactory bulb (Fig. 1A and B). The intraglomerular processes of these cells displayed high levels of TH-GFP expression, and their intertwine delimitates the glomeruli, with the soma of GFP+ cells laying around them.
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A, expression pattern of the TH-GFP transgene in the glomerular layer of the main olfactory bulb in a coronal section. GL, glomerular layer; EPL, external plexiform layer. Scale bar, 50 μm. B, magnification of the area delimitated by a dotted line in A. Scale bar, 50 μm. C, frequency distribution of the soma diameter of the cells used in this study. The distribution could be best fitted by two Gaussian curves, identifying two distinct subpopulations of cells.
Recordings with the patch-clamp technique in the whole-cell configuration were obtained from 281 DA cells in the glomerular layer. After it had been withdrawn, the pipette was always checked for the presence of fluorescence residues at the tip, as verification that the recording was actually made from a GFP+ cell.
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Cell dimensions were rather variable, as shown in Fig. 1C. Previous studies have suggested that there are two populations of DA neurones in the adult OB, based on size or location (Baker et al. 1983; Halász, 1990). In fact, the distribution of the mean cell diameter of GFP+ cells could be best fitted with two Gaussian curves, identifying two subpopulations with average sizes of 5.67 ± 0.96 and 9.48 ± 2.39 μm (R2 = 0.991); the same result could be obtained from the analysis of the membrane capacitances, whose frequency distribution could be best fitted by two Gaussians (5.41 ± 1.5 and 10.63 ± 3.45 pF, R2 = 0.975, not shown). However, we found no significant differences in the properties of the two populations.
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About 80% of DA neurones were spontaneously active. In the cell-attached configuration, action currents were recorded across the patch, usually structured in a regular, rhythmic pattern (Fig. 2A) with an average frequency of 7.30 ± 1.35 Hz (n = 31). After disruption, about 60% of the cells continued to fire spontaneous action potentials under current-clamp conditions (Fig. 2B) without any significant alteration of the firing frequency (7.84 ± 2.44 Hz, n = 14). Interspike intervals were rather constant in most of the cells (Fig. 2C), and irregular in others for the presence of sporadic misses. Occasionally, especially in isolated cells (see below), the firing was structured in bursts. We found no correlation of the firing frequency with cell size.
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A, action currents in cell-attached mode. B, action potentials in whole-cell mode. C, frequency distribution of the inter-event time for the cell shown in B. D, frequency of spontaneous firing in TH-GFP cells under the indicated experimental conditions. CA, cell attached; WC, whole cell.
This spontaneous activity was completely blocked by TTX (0.3 μM) or by Cd2+ (100 μM), but persisted after block of glutamatergic and GABAergic synaptic transmission with kynurenate (1 mM) and picrotoxin (50 μM), suggesting that it was due to intrinsic properties of the cell membrane and was not driven by external synaptic inputs.
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Occasionally we did observe spontaneous synaptic currents, which were completely blocked by a mixture of 1 mM kynurenate and 50 μM picrotoxin (not shown), and which were not further investigated for the purpose of this study.
We studied the dopaminergic neurones under current- and voltage-clamp conditions to characterize the ionic currents underlying spontaneous firing. In voltage-clamped neurones, currents were elicited both by step and ramp depolarizations.
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Depolarization activates a complicated pattern of current flow, in which a variety of conductances coexist, the most prominent of which were a fast transient sodium current and a non-inactivating potassium current (Fig. 3A and B). We identified specific ionic currents present in the cells by measurements of their voltage dependence and kinetics during step depolarizations, together with ionic substitution and blocking agents to isolate individual components of the currents. After block of the potassium currents, obtained by adding 20 mM TEA in the perfusing solution and by equimolar substitution of internal K+ ions with Cs+, a persistent inward current was observed after the fast transient inward current had completely subsided (Fig. 3C and D). The amplitude of this persistent component, measured as the average of the current amplitude during the last 10 ms of the depolarizing step, had a maximum amplitude of 223.3 ± 32.2 pA (n = 21) at –20 mV, and could be separated into two components, sustained by sodium and calcium ions (see below).
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A and B, voltage-clamp recordings from the same cell, in normal saline, held at –70 mV (A) and at –50 mV (B), and depolarized to potentials ranging from –50 to +50 mV. C, inward currents recorded under voltage-clamp conditions in response to depolarizing steps ranging from –80 to +50 mV; holding potential was –100 mV. Potassium currents were suppressed by ionic substitution of intracellular K+ ions with Cs+, and addition of 20 mM TEA in the extracellular medium. The inset shows the current–voltage relationship of the persistent inward current, averaged at the times indicated by the box. D, details of some of the traces shown in C, at higher magnification, to show the persistent inward current.
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Spontaneous activity
The presence of autorhythmic activity was the most salient feature of DA cells in the olfactory bulb, so the first efforts were aimed at elucidating the underlying mechanisms. We first tried to understand if the spontaneous activity was due to the presence of pacemaker currents or to synaptic mechanisms reverberating excitation from one cell to the other. Dissociated TH-GFP cells conserved their capacity of generating rhythmic activity, clearly indicating that this is an intrinsic property of these cells. Dissociated TH-GFP cells showed a spontaneous frequency of firing of 13.57 ± 1.79 (n = 24) and 15.75 ± 3.12 H2 (n = 14) in whole-cell and cell-attached modes, respectively (Fig. 2D). This frequency was about double the corresponding value observed in thin slices, suggesting the existence in semi-intact tissue of some inhibitory control, possibly autoinhibition, which has not been further investigated in this study.
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A likely candidate for the pacemaking process was the inward rectifier current (Ih), which we have shown to be present in a subpopulation of PG cells in a previous report (Cadetti & Belluzzi, 2001). However, we failed to observe any trace of a hyperpolarization-activated current in TH-GFP cell in the olfactory bulb (not shown), so we focused on non-inactivating inward currents.
Persistent sodium current
In DA cells, after the fast transient Na+ current had completely subsided, a persistent inward current showing no sign of inactivation after 200 ms was observed.
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We first applied TTX (0.3–1.2 μM), which suppressed a significant fraction of the persistent inward current, indicating it received a contribution from a non-inactivating, TTX-sensitive channel. This current–voltage relationship was virtually coincident with the residual persistent current measured after treatment with 100 μM Cd2+ (see below), and therefore the data were pooled. The current–voltage relationship of the fraction of current abolished by TTX or remaining after Cd2+ treatment is shown in Fig. 4A. The persistent sodium current, INa(P), was activated at potentials more negative than –70 mV, and reached a maximum amplitude of –27.5 ± 2.97 pA at –30 mV. The corresponding conductance–voltage relationship, calculated by dividing the current amplitude by the sodium driving force, could be fitted by a Boltzmann equation with a midpoint at –48.8 mV and a slope of 6.51 mV nS–1 (Fig. 4B). The maximum value of gNa(P) conductance was 0.41 nS, about 200 times smaller than that of the fast sodium current (INa(F), see below), but contrary to the latter, this current is activated in the pacemaker range, showing an amplitude of –7.3 pA at –60 mV.
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A, I–V relationship. Pooled data obtained as fraction of non-inactivating current suppressed by TTX (n = 12) and residual persistent current after Cd2+ block (n = 6). B, conductance–voltage relationship of persistent sodium current, obtained from the mean data shown in A. The continuous curve is the Boltzmann fit, with upper asymptote of 0.41 nS, midpoint at –48.8 mV and slope of 6.51 mV nS–1.
The calcium currents
After block of the TTX-sensitive component and suppression of the K+ current by equimolar substitution of intracellular K+ with Cs+, a persistent inward current could be observed at the end of prolonged depolarizing steps (Fig. 5A, upper traces). This residual fraction could be almost completely blocked by Cd2+ or Co2+ ions (Fig. 5A, lower traces), suggesting that this second component was sustained by calcium ions. The very small fraction of current remaining after TTX and Cd2+ block has not been further investigated in the present study.
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A, calcium current recorded in response to depolarizing voltage steps to potentials ranging from –70 to +10 mV from a holding potential of –100 mV. Above: tracings recorded in the presence of 1.2 μM TTX, 20 mM TEA in the extracellular solution, and with Cs+ as a substitute for K+ ions in the intracellular solution. Lower tracings were recorded under the same conditions after addition of 100 μM Cd2+. B, barium currents recorded in the same conditions described for A. C, I–V relationship of Ca2+ and Ba2+ currents from a 10 neurone sample from thin slices. D, activation time constant, measured from a 10 neurone sample by fitting a single exponential to the rising phase of the current. The continuous line is described by the equation: aCa(L) = 1.23 + exp(–V/74.6). E, effect of 10 μM nifedipine on calcium current in a group of six TH-GFP PG cells in slices. F, histogram showing the effect of nifedipine (10 μM, cells shown in E) and calcicludine (1 μM, not shown, n = 7) measured at –10 mV.
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Using classical pharmacological tools, ionic substitutions and voltage-clamp protocols, we could dissect the voltage-dependent Ca2+ currents, Cav, into several components, including both high-voltage activated (HVA) and low-voltage activated (LVA) currents.
The larger of these components, by its overall kinetics, its voltage dependence and the absence of inactivation, was identified as a possible L-type Ca+ current. Its properties were studied in slices, after blockage of the Na+ currents with 0.3–1.2 μM TTX and of the K+ currents by equimolar substitution with Cs+ in the pipette-filling solution and 20 mM TEA in the perfusing bath. The protocols used were either voltage steps or voltage ramps, giving virtually identical results. The I–V relationship of the Ca+ current (Fig. 5C), measured in a 10 neurone sample averaging the last 5 ms at the end of a 40 ms depolarizing step, had a maximum amplitude of –108.7 ± 11.9 pA at –10 mV. The corresponding conductance–voltage relationship showed a maximum conductance of 2.3 nS, with a midpoint at –25.6 mV.
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Equimolar substitution of Ca2+ with Ba2+ increased the amplitude of this current by a factor of about 3 (Fig. 5C), without changing the time constant of activation. On this current we tested the effects of two blockers of L-type calcium channels, nifedipine and calcicludine. The fraction of current blocked by the two drugs at different voltages was quantified by subtraction of I–V data acquired before and after treatment.
The effects of 10 μM nifedipine on peak Ca2+ current amplitude was assessed in six PG cells (Fig. 5E and F). On average, the drug blocked 61.1 ± 14% of the current measured at the point of its maximum amplitude (–10 mV).
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A 60 amino acid peptide, calcicludine (CaC), has been described as having a powerful effect on all type of high-voltage-activated Ca2+ channels (L-, N- and P-type) (Schweitz et al. 1994). Since one of the regions of the CNS presenting the highest densities of 125I-labelled CaC binding sites is the glomerular layer of the olfactory bulb (Schweitz et al. 1994), we tested the ability of this toxin in suppressing the non-inactivating Ca2+ current found in the DA neurones. CaC (1 μM) was much more effective than nifedipine, with an inhibitory action averaging 72.7 ± 3.13%.
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We also tried to define whether other types of HVA neuronal Cav channels (P/Q-, N-) were present in TH-GFP cells. Using classical blockers, like -conotoxin GVIA (0.82 μM) that blocks the N-type, or spider toxin -agatoxin IVA (10 nM) that blocks P/Q-type channels, we observed the suppression of a fraction of HVA Ca2+ current remaining after nifedipine block (at –10 mV 38 and 42%, respectively, not shown), suggesting the presence of limited amounts of the corresponding HVA Ca2+ channels.
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Since the long-lasting HVA Cav currents were not directly involved in the pacemaker process (see below), and for the dominance of L- over N- and P/Q-type current, for the purpose of the numerical reconstruction of the electrical activity of these cells (see below), they were kinetically modelled as a unique, non-inactivating component. The rising phase of the current was fitted by a single exponential, with a time constant of 1.2 ± 0.3 ms at 0 mV (n = 10, see legend of Fig. 5D for further details).
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Since in situ hybridization experiments have localized the expression of two transcripts (1G and 1I) of the T-type calcium channel in the glomerular layer of the olfactory bulb (Talley et al. 1999; Klugbauer et al. 1999), we checked for the presence of LVA Ca2+ current. Unfortunately several characteristics of these channels hampered their study in our preparation. First, contrary to the cardiac cells or transfected cells, in TH-GFP interneurones this current is small: we have calculated a maximum conductance of 0.35 nS, corresponding to a peak current of about 20 pA at –35 mV. The problem was further complicated by the fact that on one hand it was difficult to get accurate space clamping in slices, and on the other hand, in isolated cell preparations the current was difficult to resolve, probably because of the preferential localization of these channels on the dendrites (Perez-Reyes, 2003). Second, the conductance of these channels cannot be significantly increased by substitution of Ca2+ with Ba2+ ions, as it can be done with HVA channels. Third, there are no effective pharmacological tools for the study of T-type Ca2+ channels, because they are relatively resistant to most organic calcium channel blockers, such as dihydropyridines, that block the L-type, or peptide toxins, such as -conotoxin or -agatoxin.
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Despite these difficulties, we succeeded in isolating a T-type calcium current in dissociated cells (Fig. 6). The protocol used was a rapid ramp (7 V s–1) from –100 to 40 mV, in the presence of TTX (1 μM) and after substitution of Ca2+ with Sr2+, which is known to have a slightly higher permeability than Ca2+ in T-type Ca2+ channels (Takahashi et al. 1991). Under these conditions, an inward inflection peaking at about –40 mV, distinct from the peak due to the L-type Ca2+ channels (Fig. 6A), could be seen. Nickel is a non-selective inhibitor of calcium channels, but transient low voltage-activated (LVA) T-type Ca2+ channels are particularly sensitive (IC50 < 50 μM) (Perchenet et al. 2000; Wolfart & Roeper, 2002), whereas other HVA Cav channels (L-, P/Q and N-type) are less sensitive (IC50 > 90 μM) (Zhang et al. 1993; Randall, 1998). In fact 100 μM nickel did selectively eliminate the first peak, leaving the second unaltered. The difference, averaged in three cells, is shown in Fig. 6B, and the conductance, calculated assuming a Ca2+ equilibrium potential of 45 mV, is illustrated in Fig. 6C. The current activates at potentials positive to –65 mV and peaks at about –35 mV, with a maximum conductance of 0.35 nS. The point of half-activation is –45.3 mV, which is in line with the known values for this current (Perez-Reyes, 2003).
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A, voltage-clamp ramps were performed from –80 to +40 mV at a speed of 7 V s–1 in the presence of TTX (1 μM), and after substitution of Sr2+ for Ca2+; the traces were corrected for leakage. Under these conditions, a distinct bump can be seen at –40 mV, preceding the HVA calcium current (here peaking at –10 mV), which is selectively suppressed by 100 μM Ni2+. B, transient Ca2+ current generated using the protocol described in A calculated by subtracting the current–voltage curves in control and in the presence of 100 μM Ni2+. The low-Ni2+ sensitive current (average from three cells) is activated at membrane potentials more positive than –60 mV. C, conductance–voltage relationship of the low-Ni2+ sensitive current. The continuous line is a Boltzmann fit, with a midpoint at –45.3 mV and a maximum conductance of 0.35 nS. All recordings in this figure were performed from spontaneously bursting dissociated TH-GFP cells.
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The pacemaker currents
We next tried to elucidate the ionic basis of the pacemaker current underlying the spontaneous firing. We first found that the Ca2+ current is involved in the pacemaker process, as 100 μM Cd2+ completely and reversibly blocked the spontaneous firing (Fig. S1A and B in Supplemental material). Then, using a panel of different Cav channels inhibitors, we tried to define which types of neuronal Cav channels (L-, P/Q-, N-, R- and T-type) contributed to the pacemaker current.
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The classical selective L-type Ca2+ channel antagonist nifedipine (10 μM), which blocked the long-lasting Ca2+ current by about two thirds (Fig. 5E and F), had no effect at all in the spontaneous firing frequency, either in cell-attached mode (Fig. S1C in Supplemental material) or in whole-cell configuration, in a total of six cells recorded in slices. Also calcicludine (1 μM), a powerful although less selective HVA channel blocker (Schweitz et al. 1994), which inhibited the long-lasting Ca2+ channel component (73%, Fig. 5F), was equally ineffective, even after very long periods of application (Fig. S1D and E in Supplemental material). Analogous results were obtained using other classical blockers of HVA Ca2+ channels, like -conotoxin GVIA (0.82 μM), which blocks the N-type, or spider toxin -agatoxin IVA (10 nM) which blocks P/Q-type channels. Both did suppress a fraction of the residual HVA Ca2+ current after nifedipine block (at –10 mV 38 and 42%, respectively), suggesting the presence of the corresponding types of HVA Ca2+ channels, but none of them affected the spontaneous firing (not shown).
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Among the remaining candidates for a role in pacemaking was the LVA, T-type channel. Mibefradil has been reported to inhibit T-type calcium channel current in several neuronal types (Randall & Tsien, 1997; Viana et al. 1997; Todorovic & Lingle, 1998). We therefore used this drug, which proved to be considerably powerful in blocking the spontaneous activity of PG DA neurones, both in cell attached and in whole-cell mode (Fig. S2A and B in Supplemental material). Mibefradil (5–10 μM) completely and reversibly blocks the spontaneous activity, inducing an evident hyperpolarization that on average amounted to about 15 mV (Fig. S2B). Nickel (100 μM), that we have shown to be a selective blocker of T-type calcium current in these cells (Fig. 6A), induced a reversible block of spontaneous firing, also accompanied by a hyperpolarization of 10–15 mV (Fig. S2C), an effect almost superimposable on that observed with mibefradil, further confirming a role of ICa(T) in the pacemaking process.
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Isolation and kinetic characterization of the fast transient Na+ current
A classical fast transient, TTX-sensitive sodium current was present in all the DA neurones studied. The current was isolated by blocking the Ca2+ current with 100 μM Cd2+, and by equimolar substitution of intracellular K+ with Cs+; in addition, the K+ channels were blocked by adding 20 mM TEA in the perfusing solution (and occasionally also in the intracellular solution to complete the blockade). On this current we performed a standard Hodgkin-Huxley-type analysis (Hodgkin & Huxley, 1952), and the relevant results are summarized in the Appendix. Representative recordings, with an explanation of the protocols used, are shown in the Supplemental material (Fig. S3, and relative legend).
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Isolation and time course of the potassium currents
A delayed rectifier-type potassium current was present in TH-GFP cells (e.g. Figure 3B) which has been kinetically characterized. The current, isolated by blocking the sodium current with TTX, was calcium dependent only for a small part (at 0 mV the fraction suppressed by Cd2+ 100 μM was about 10%), and thus it has been modelled as a single component. The equations describing the time- and voltage-dependence of the potassium current are given in the Appendix and illustrated in Fig. S4 (in Supplemental material).
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Modelling the natural burst firing in bulbar DA neurones
Finally, we have modelled the bulbar DA neurones in Hodgkin-Huxley terms (Hodgkin & Huxley, 1952), considering the cell as a single electrical and spatial compartment. As for the conductances considered, we incorporated the two sodium currents (fast transient and persistent), the L-type Ca2+ current, the delayed rectifier K+ current, all according to the experimental data presented above, and the T-type calcium current. Since our characterization of this current was incomplete (and because of the difficulty in obtaining a complete kinetic description of this current), in our model we have integrated our data on the activation process with others, concerning the inactivation, derived from the literature (Wang et al. 1996; Perez-Reyes, 2003), and consistent with our experimental data.
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All the equations and parameters used, as well as the assumptions made, are listed in the Appendix. The solution of the set of differential equations describing the kinetics of the currents considered lead to the tracings reported in Fig. 7. The model we set up shows intrinsic spiking capabilities with full-size action potentials at the same frequencies observed in TH-GFP cells.
A, voltage tracings. B, current tracings including the five conductances: INa(F), INa(P), ICa(T), ICa(L), IK(V). C and D, enlargement of the last two events of A and B to show the pacemaking process. In D the outward current has been omitted, and the inward currents have been amplified by a factor of about 500 with respect to B.
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The model is crucial to understand the interplay of the currents underlying the pacemaking process. As it can be clearly seen in Fig. 7C, during the interspike interval, the currents which cause the progressive depolarization of the cell are, in order, the T-type calcium current, and then the persistent sodium current. Although these currents are amazingly small in amplitude (max. 4 pA) compared with fast transient sodium and delayed rectifier potassium currents associated to the action potential (about 1 nA), they are nevertheless sufficient to depolarize these cells, due to their rather high input resistance (about 700 M).
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It is the T-type calcium current which sets in motion the depolarizing process but, both INa(P) and ICa(T) are necessary to sustain spontaneous firing as the selective block of one or both abolish spontaneous activity: the model cell is still capable of responding with a single action potential to the injection of a depolarizing current pulse, but it fails to fire repetitively. The T-type calcium conductance amplitude is critical in determining the firing frequency: small changes in its value (from 0.35 to 0.4 nS) are sufficient to drive the intrinsic spiking from 8 to 16 Hz.
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The model thus confirms that, in addition to INa(P), another component is necessary to sustain repetitive firing: a T-type calcium current. Together with the experimental finding that treatments which block ICa(T), such as mibefradil and nickel at micromolar concentrations, are both capable of preventing repetitive firing, it therefore appears that this current is an essential component of the pacemaking process.
Discussion
This study represents the first description of the functional properties of DA neurones in the mammalian olfactory bulb. The animal model used for these experiments, a strain of transgenic mice expressing a reporter protein under the TH promoter (Sawamoto et al. 2001; Matsushita et al. 2002), allows an easy identification of DA neurones both in thin slices and dissociated cells, proving to be a superb tool for targeting live DA neurones in electrophysiological studies. The main results obtained are the demonstration that DA neurones in the OB are autorhythmic, and the description of the interplay of subthreshold currents underlying intrinsic spiking.
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Distribution and general properties of DA neurones
In mammalians, neurones expressing high levels of the reporter protein were found in the glomerular layer, as expected from abundant literature, indicating that this is the only region of the main olfactory bulb where TH is expressed (Halász, 1990; Kratskin & Belluzzi, 2003). A previous study in the same mice strain has demonstrated an overlapping expression of the fluorescent reporter and TH protein in olfactory bulb only in those DA neurones that received afferent stimulation from receptor cells (Baker et al. 2003).
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DA neurones in the mice OB have a complement of voltage-dependent currents, which have been kinetically characterized in this study. Among these, the persistent Na+ current deserves some comment. Many neurones in the mammalian CNS have a non-inactivating component of the TTX-sensitive sodium current (Crill, 1996). Although its magnitude in bulbar DA neurones is about 0.5% of the transient sodium current, INa(P) appears to have an important functional significance because it is activated at potentials 8–10 mV more negative than the transient sodium current. At this potential few voltage-gated channels are activated and the neurone input resistance is high. The conductance–voltage relationship for gNa(P) in TH-GFP DA neurones has a half-activation point at –47.7 mV, very close to the values found in hippocampal CA1 neurones (French et al. 1990) and pyramidal neurones of the neocortex (Brown et al. 1994). Although this current might appear small, 7.3 pA at –60 mV (Fig. 4A), it suffices to depolarize the cell membrane of these cells, which have an average input resistance of 700 M, by about 5 mV.
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We have considered the possibility that INa(P) is in fact a window current, the steady current predicted by the HH model and arising from overlap of the steady-state activation and inactivation curves for sodium conductance (Attwell et al. 1979; Colatsky, 1982). Based on the kinetic analysis of the fast Na+ current (Supplemental material, Fig. S3H), we calculated the window current at different potentials. The numerical simulation indicates this current does provide a measurable contribution to the TTX-sensitive persistent inward current. However, this contribution, which is maximal at –45 mV, falls to virtually zero at –30 mV, the potential at which the persistent sodium current displays its maximum amplitude (Fig. 4A). In other words, INa(P) and the window current of INa(F) develop at different potentials, and therefore are distinct currents.
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Interaction of ionic currents to produce spontaneous firing
The pharmacological treatments, ion substitution experiments, kinetic analysis and numerical simulations, allow a rather precise understanding of mechanisms underlying spontaneous firing in TH-GFP cells. The first observation is that the slow depolarization between spikes is sustained by the persistent, tetrodotoxin-sensitive sodium current and by the T-type calcium current, without any intervention of hyperpolarization-activated, h-type current. This is of some interest, as the h-current has been described in cells of the glomerular layer (Cadetti & Belluzzi, 2001). However, if it is true that in some dopaminergic midbrain neurones the h-channels have been shown to be actively involved in pacemaker frequency control (Seutin et al. 2001; Neuhoff et al. 2002), the same has not been observed in many other dopaminergic neurones, as in the retina (Feigenspan et al. 1998), or in the ventral tegmental area (Neuhoff et al. 2002).
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The role of a calcium current in rhythm generation is revealed by the rapid and reversible block of the spontaneous firing by Cd2+, both in slices and in dissociated cells (Supplemental material, Fig. S1B). However, among the many HVA Ca2+ channels present in the DA neurones of the olfactory bulb (a large L-, and smaller N- and P/Q types), none proved to be effective in the control of spontaneous firing. On the contrary, conditions which are known to block the LVA T-type Ca2+ channels (mibefradil and nickel in the micromolar range) did break off the spontaneous activity.
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To better understand the interplay of conductances underlying the spontaneous activity, we developed a numerical HH-type model (Hodgkin & Huxley, 1952) of TH-GFP cells, based on the kinetic characterization of the voltage-dependent currents described above, which reproduces their behaviour fairly well. This model is useful not only for the validation of measurements and analysis, but, first and foremost, because it provides an in-depth quantitative description of the events underlying the pacemaking process (Fig. 7C).
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Both INa(P) and ICa(T) are necessary to sustain spontaneous firing as the selective block of one or both abolish spontaneous activity. However, it is the T-type calcium current which sets in motion the depolarizing process: although essential to the rhythm generation, INa(P) replaces ICa(T) only in the second half of the depolarizing phase.
Small changes in the parameters of INa(F) and IK(V), such as the half-activation point shift of a few millivolts, were sufficient to arrest spontaneous firing but did not affect the capacity to respond with single action potentials to depolarizing stimuli. This proves that the pacemaking process is due to an interplay of conductances which are in a delicate and precise equilibrium and, furthermore, it confirms that the model is capable of capturing the essential features of the excitability profile of these cells.
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In conclusion, the experimental observation that the block of ICa(T) with mibefradil or nickel at micromolar concentrations are both capable of preventing repetitive firing are well explained by the model, which assigns to this current the role of primum movens in the pacemaking process.
Significance to olfactory function
Our results, and especially the indication that DA neurones in the glomerular layer are autorhythmic, open the field to many speculations.
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As indicated by an abundant literature, dopamine is inhibitory in the olfactory bulb, acting primarily on presynaptic D2 receptors located in the olfactory nerve terminal (Brünig et al. 1999; Hsia et al. 1999; Wachowiak & Cohen, 1999; Berkowicz & Trombley, 2000; Ennis et al. 2001; Davila et al. 2003). This inhibitory role has been observed both in mammalians and in the frog olfactory bulb, although in the latter both anatomical localization of D2-like receptors and functional data on dopamine involvement in information processing differ in several aspects from those reported in mammals (Duchamp-Viret et al. 1997; Davison et al. 2004).
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In mammalian systems, it is known that the glomerular neuropile, far from being a homogeneous structure, shows a complex subcompartimental organization: within a single glomerulus, olfactory nerve (ON) islets delimit areas in which dendritic branches receive sensory input from ON terminals, and are well separated from non-ON zones, from which ON terminals are excluded (Chao et al. 1997; Kasowski et al. 1999; Toida et al. 2000). Some TH-immunoreactive PG cells arborize on the ON islets, others in the non-ON zone (Kosaka et al. 1997). Although it has been reported that in some mouse strains TH-positive cells are not in contact with ON terminals (Weruaga et al. 2000), in the strain used for these experiments – one of the most common (C57BL/6 J) – such contacts have been demonstrated (T. Kosaka and K. Kosaka, personal communication).
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The glomerular compartmentalization supports the hypothesis that information processing is subdivided regionally within the mammalian glomerulus (Kasowski et al. 1999). DA neurones establishing contacts in ON- or in non-ON zones, possibly play different roles. Within the ON zones, dendrites of DA neurones receive excitatory synapses from ON axon terminals (Chao et al. 1997; Kasowski et al. 1999; Toida et al. 2000). It has been reported that D2 dopaminergic receptors are located in ON terminals (Levey et al. 1993; Coronas et al. 1997), and electrophysiological studies have shown that their activation can reduce the probability of glutamate release, and hence the excitation of projection neurones (Duchamp-Viret et al. 1997; Hsia et al. 1999; Berkowicz & Trombley, 2000; Ennis et al. 2001; Davison et al. 2004). In fact, the most significant impact of DA neurones is expected at the level of the synaptic triad formed by the ON and the dendrites of mitral/tufted (MT) cells and PG cells, where DA neurones directly control the input of projection neurones from receptor cell axons (Brünig et al. 1999). Since the ON islets appear to be further segregated from the rest of the glomerular neuropile due to the presence of ‘glial wraps’ (Kasowski et al. 1999), the spontaneous activity of DA neurones (which implies continuous release of dopamine within a restricted space), would create a condition of tonic inhibition of the ON. The situation in the frog olfactory bulb appears entirely different, in that DA neurones directly inhibit the projection neurones (Duchamp-Viret et al. 1997; Davison et al. 2004).
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In addition, DA neurones send their dendrites also into non-ON zones, where they contact dendrites of projection neurones and interneurones, and centrifugal axons. Projection neurones express D1 and D2 receptors (Brünig et al. 1999; Davila et al. 2003), and it has been shown that dopamine exerts a complex modulatory action between them and interneurones (ibidem), an effect that also in this case would be amplified by the restricted space of the glomerular neuropile. Within this framework, dopamine might play a central role in the processing of olfactory information by acting at two levels: it would control the input of the sensory signal, and it would modulate the mechanism of GABAergic inhibition (Brünig et al. 1999; Davila et al. 2003).
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It remains to be explained why DA neurones in the glomerular region of the OB are among the very few in the mammalian CNS which are generated also in adulthood. This raises a series of questions concerning the mechanisms controlling their migration and differentiation, and, also, it opens interesting perspectives for the exploitation of the olfactory bulb as a source of undifferentiated DA cells that could be expanded ex vivo and used for transplants in neurodegenerative diseases.
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Supplemental material
The online version of this paper can be accessed at:DOI: 10.1113/jphysiol.2005.084632
http://jp.physoc.org/cgi/content/full/jphysiol.2005.084632/DC1
and contains supplemental figures.
This material can also be found at:http://www.blackwellpublishing.com/products/journals/suppmat/tjp/tjp821/tjp821/sm.htm
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Cable properties
Input resistance: 706 M; membrane capacitance 6.7 pF
Whole-cell conductances (nS)
Equilibrium potentials (mV)
Fast transient sodium current – INa(F)
Steady-state activation:
Steady-state inactivation:
, http://www.100md.com Activation time constant:
Inactivation time constant:
De-inactivation time constant: 42 ms
Delayed rectifier potassium current – IK(V)
Steady-state activation:
Activation time constant:
Deactivation time constant:
T-type calcium current – ICa(T)
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Steady-state activation:
Steady-state inactivation:
Activation time constant:
Inactivation time constant:
Persistent sodium current – INa(P)
Steady-state activation:
Activation time constant: 0.9 ms
L-type calcium current – ICa(L)
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Steady-state activation:
Activation time constant:
Deactivation time constant: 0.587 ms
The reconstruction has been made using time increments of 83.3 μs, corresponding to a frequency of 12 kHz.
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