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INNOVATIVE METHODOLOGY
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts
Submitted 2 March 2005; accepted in final form 15 July 2005
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Pinching and Powell 1971a,b; Shepherd and Greer 1998).
The electrical properties and functional roles of periglomerular cells are not well understood. Periglomerular cells represent a heterogeneous class of neurons, and at least three distinct groups can be recognized on the basis of their immunoreactivity for glutamic acid decarboxylase (GAD), calbindin, and calretinin (Kosaka et al. 1995, 1998). Periglomerular cells are also likely to be functionally heterogeneous because they exhibit different K+ conductances (Puopolo and Belluzzi 1998a) as well as different firing behaviors (Hayar et al. 2004; McQuiston and Katz 2001). Various groups of periglomerular cells target different domains within the glomerulus with only some of them receiving synaptic input from the axons of the olfactory nerve (Kasowski et al. 1999; Kosaka et al. 1998; Toida et al. 2000).
Understanding the functional role of periglomerular cells depends on the ability to reliably distinguish individual populations of the neurons. Among the periglomerular cells that contain GAD or GABA, some also express tyrosine hydroxylase (Gall et al. 1987; Halasz et al. 1981; Kosaka K et al. 1995; Kosaka T et al. 1985), the rate-limiting enzyme for the synthesis of catecholamines, and thus most likely release dopamine as a modulatory transmitter. By using a transgenic line of mice in which catecholaminergic (CA) neurons in the CNS express human placental alkaline phosphatase (PLAP) under the control of the promoter of the tyrosine hydroxylase gene (Gustincich et al. 1997), we were able to identify a population of dopaminergic periglomerular cells after acute dissociation of the main olfactory bulb. Current-clamp recordings showed that these neurons spontaneously generate action potentials in a rhythmic fashion. Voltage-clamp recordings then allowed us to explore the ionic currents responsible for their pacemaker activity.
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METHODS |
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Transgenic mice were used in which CA neurons in the CNS express human PLAP on the outer surface of the cell membrane (Gustincich et al. 1997). Mice were 12–17 days old; development, migration, and synapse formation by periglomerular cells appear essentially complete at these ages (Brunjes and Frazier 1986; McLean and Shipley 1988). Anesthesia was carried out by intraperitoneal injection of 0.1 ml of a solution containing 5% ketamine HCl (Ketaset; Fort Dodge Laboratories, Fort Dodge, IA) and 1% xylazine (Rompun; Bayer, Shawnee Mission, KS). The procedure to release isolated dopaminergic periglomerular cells was adapted from methods used previously for other types of central neurons (Do and Bean 2003; Raman and Bean 1999). After decapitation, the brain was removed and placed in ice-cold dissociation medium (DM) containing (in 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. The olfactory bulbs were removed from the brain and cut into 400-µm horizontal slices with a vibrating tissue slicer (DTK-1000, DSK, Dosaka, Japan). Slices containing the glomeruli were incubated in DM containing 3 mg/ml protease type XXIII (Sigma, St. Louis, MO) for 30–40 min at 35°C. After enzymatic digestion, slices were washed with DM containing 1 mg/ml bovine serum albumin (Sigma) and 1 mg/ml trypsin inhibitor (Sigma). The enzyme-treated slices were then gently triturated through a series of fine-bore, fire-polished Pasteur pipettes to generate a suspension of isolated neurons. The dissociated neurons were directly plated on the glass bottom of concanavalin A-coated (1 mg/ml) recording chambers and kept at 37°C in 5% CO2-95% O2 for 
1 h before commencement of recordings. Dopaminergic neurons were identified by labeling of their membrane with a monoclonal antibody to PLAP (De Groote et al. 1983) directly conjugated to the fluorochrome Cy3 (E6-Cy3). Techniques for labeling of tissue sections (Fig. 1) followed those of Gustincich et al. (1997).
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Equipment and recording techniques were described previously (Puopolo et al. 2001). Briefly, E6-Cy3-stained dopaminergic neurons were identified by scanning the coverslip in epifluorescence. The remaining procedures were carried out in visible light with Nomarski optics. It is unlikely that neurons are damaged as a result of expression of PLAP, immunolabeling, or fluorescence because in other experiments, we have recorded from dopaminergic cells in the substantia nigra pars compacta using the same transgenic mouse and labeling procedure and saw no difference in electrophysiological properties or cell viability compared with extensive recordings from unlabeled dopaminergic neurons in wild-type mice.
Current and voltage traces were low-pass-filtered at 2 kHz using the internal Bessel filter of the amplifier (Axopatch 200B, Axon instruments, Foster City, CA). The sample frequency was 20–50 kHz for whole cell voltage-clamp recordings and 10 kHz for current-clamp recordings. The resistance of the patch pipettes was 3–5 M
after filling with the internal solution. In voltage-clamp experiments, the series resistance of the pipettes was 10–15 M and could be compensated 
80% after cancellation of capacitative transients. Current-clamp recordings were carried out in the fast current clamp mode to reduce distortion of action potentials (Magistretti et al. 1996, 1998).
Unless noted otherwise, the extracellular bath solution for current-clamp recordings contained (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose. The intracellular solution contained (in mM) 120 K-gluconate, 6 NaCl, 0.5 CaCl2, 4 MgCl2, 2 Mg-ATP, 0.3 Na-GTP, 14 phosphocreatine, 5 EGTA, 10 HEPES and 10 glucose. Sodium and Ca2+ currents were obtained by subtraction: for the Na+ current, the extracellular solutions contained (in mM) 137 NaCl, 1.8 CoCl2, 1 MgCl2, 5.4 TEA-Cl, 10 4-aminopyridine, 5 HEPES and 10 glucose with and without 1 µM tetrodotoxin (TTX). For determining Ca2+ current, 1.8 CoCl2 was substituted for CaCl2 in an extracellular solution containing (in mM) 137 NaCl, 1.8 CaCl2, 1 MgCl2, 5.4 TEA-Cl, 10 4-aminopyridine, 5 HEPES, and 10 glucose and 1 µM TTX.
A stock solution of TTX (Research Biochemicals, Natick, MA) was made in 2 mM citric acid and stored at –20°C. Drugs were diluted to final concentration in the extracellular solution before the experiment and applied to single cells in the external bath solution by gravity flow through an array of microcapillary tubes controlled by a rapid solution changer (RSC-200, Biologic, Claix, France). Data were acquired with pClamp 8 (Axon instruments), and data analysis was carried out using Clampfit 8 (Axon instruments) and IGORPro version 3.14 (WaveMetrics, Lake Oswego, OR). Reported voltages were corrected for the junction potential between the K-gluconate-based internal solution and the extracellular solution [–11 mV, measured using a flowing 3 M KCl electrode as described in Neher (1992)]. Data are presented as means ± SD. All experiments were carried out at room temperature (22–24°C).
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RESULTS |
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To identify dopaminergic periglomerular cells, we used a transgenic mouse line (Gustincich et al. 1997) in which placental alkaline phosphatase (PLAP) is expressed under the control of the promoter sequence of the gene for tyrosine hydroxylase (Banerjee et al. 1992). Figure 1 illustrates the location in the main olfactory bulb of the neurons that express PLAP and tyrosine hydroxylase. The staining for both proteins was very intense in the glomerular layer (Fig. 1A). PLAP (Fig. 1B) and tyrosine hydroxylase (C) were both expressed in two different cell types. The vast majority of the positive neurons were periglomerular cells, 7–10 µm in diameter, situated at the periphery of the olfactory glomeruli. In addition, a small number of external tufted cells were stained in the external plexiform layer. Because PLAP resides on the outer surface of the cell membrane, living neurons that express tyrosine hydroxylase could be fluorescently labeled by a monoclonal antibody to PLAP directly conjugated to Cy3 after neurons were dissociated from the olfactory bulb (Fig. 1B, inset). After dissociation, labeled cells fell into two distinct populations. The majority had small perikarya (7–10 µm) and cell capacitances of 5–10 pF, whereas a minority had perikarya of 13–15 µm and capacitances of 15–20 pF. These populations of two different sizes correspond to what would be expected of dopaminergic periglomerular cells and dopaminergic external tufted cells, respectively (Hayar et al. 2004; Puopolo and Belluzzi 1998a,b; Pignatelli et al. 2005). Therefore to confine our study to periglomerular cells, we performed experiment exclusively on cells with cell body diameters <10 µm. Of the cells considered in this study, average diameter was 8.6 ± 1.0 µm (n = 20) and average capacitance was 7.8 ± 2.0 pF (n = 72).
Spontaneous firing in dopaminergic periglomerular cells
Solitary dopaminergic periglomerular cells were found to be spontaneously active. Spontaneous activity was evident as action currents on formation of seals using patch pipettes in the cell-attached mode (Fig. 2A), and firing was highly regular and rhythmic (B). After breaking the cell membrane and switching to whole cell current-clamp mode, the majority of cells (77%) continued to fire spontaneously (Fig. 2, C and D). Occasionally, neurons fired spontaneously in cell-attached configuration but were silent in whole cell-current clamp apparently due to depolarization block; these cells were not used for further analysis. The average rate of firing was 8.2 ± 4.6 Hz (n = 20). The spontaneous firing had full-blown action potentials with an average amplitude of 85 ± 12 mV (n = 20), measured from an average after-spike potential of –63.5 ± 5.9 mV (n = 20). The width of action potentials (measured at half-amplitude) was 2.1 ± 0.8 ms (n = 20). Cells had an average input resistance of 1.4 ± 0.5 G (n = 26).
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The ability of isolated dopaminergic periglomerular cells to fire action potentials spontaneously demonstrates that such activity is intrinsic to the neurons and does not require synaptic inputs or modulatory transmitters or peptides. In this respect, dopaminergic periglomerular cells resemble midbrain dopaminergic neurons (Grace and Bunney 1984; Hainsworth et al. 1991) and retinal dopaminergic amacrine cells (Feigenspan et al. 1998). In some cells that are spontaneously active, including sinoatrial myocytes of the heart (DiFrancesco et al. 1986), thalamic relay neurons (McCormick and Pape 1990), and some midbrain dopaminergic neurons (Neuhoff et al. 2002), the hyperpolarization-activated cation current known as Ih appears to play a primary role in driving the spontaneous depolarization that leads to spike generation. To test the possible contribution of Ih to pacemaking, dopaminergic periglomerular cells were challenged with the blocking agent ZD 7288 (Gasparini and DiFrancesco 1997). Applied at a concentration of 30 µM, ZD 7288 had little effect on spontaneous activity (Fig. 3). In results collected from four dopaminergic periglomerular cells, the drug caused a small depolarizing shift of the membrane potential, from –74 ± 2 to –73 ± 2 mV (measured at the trough), and a small increase of the firing frequency, from 8.2 ± 2.4 to 9.0 ± 2.4 Hz. Both changes are the opposite of what one would expect from blocking the inward-directed Ih. These results suggest that Ih plays little or no role in pacemaking. The modest depolarization and speeding of firing most likely result from small nonspecific blocking effects of ZD 7288 on K+ currents. We also tested the effect of 1 mM Cs+, another blocker of Ih. Cesium also had little effect on spontaneous firing rate (8.5 ± 1.4 Hz in control, 8.7 ± 1.4 Hz with 1 mM Cs+, n = 5) or on membrane potential (–65 ± 6 mV in control, –64 ± 7 mV in Cs+), again consistent with little activation of Ih.
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Another candidate for an inward ionic current driving spontaneous firing is voltage-activated Ca2+ current. Blocking various voltage-dependent Ca2+ currents halts or slows pacemaking in some catecholaminergic neurons, including dopaminergic neurons of the substantia nigra (Kang and Kitai 1993; Mercuri et al. 1994; Nedergaard et al. 1993). In midbrain dopaminergic neurons, a component of Ca2+ current activates at potentials as negative as –60 to –50 mV, well-positioned for a role in pacemaking (Durante et al. 2004; Wilson and Callaway 2000). To test the possible contribution of Ca2+ current to spontaneous firing, dopaminergic periglomerular cells were challenged with a solution in which Ca2+ ions were replaced by equimolar (1.8 mM) Co2+. This treatment stopped spontaneous firing, and the cessation of firing was accompanied by a hyperpolarization (Fig. 4A), a result consistent with block of an inward current. These results were obtained in each of five neurons tested, with cessation of rhythmic firing and hyperpolarization of the cell (from –66 ± 2 mV in control to –73 ± 4 mV with Co2+, n = 5). The cessation of pacemaking after Co2+ substitution for Ca2+ was not due to a generalized or nonspecific loss of excitability because in some of the cells there were occasional, rare, sporadic action potentials of full height in the Co2+ solution.
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; Williams et al. 1984). Such behavior is seen in some dopaminergic midbrain neurons, suggestive that pacemaking may be driven by an underlying electrical oscillation due to Ca2+ current (Wilson and Callaway 2000). We tested for this behavior in dopaminergic periglomerular cells. In these neurons, however, application of TTX most commonly (10 of 13 cells) produced complete quiescence accompanied by a net hyperpolarization (Fig. 4B). The effect of TTX was fully reversible, and spontaneous firing at the original frequency resumed after washing TTX for 50–60 s. The hyperpolarization that accompanies cessation of firing with TTX application suggests that in addition to supporting spikes, TTX-sensitive Na+ channels contribute subthreshold current that flows during the interspike interval. In 3 of 13 cells, there were small oscillations in membrane potential in TTX, reminiscent of the Ca2+-dependent oscillations in midbrain dopamine neurons (Wilson and Callaway 2000) but far smaller (4.2 ± 2.5 mV, n = 3) and less regular. Thus although blocking Ca2+ current stops spontaneous activity in dopaminergic periglomerular neurons, the mechanism of pacemaking seems different from in midbrain dopamine neurons, where Ca2+ currents appear to drive pacemaking by producing an underlying large-amplitude voltage oscillation that persists when spikes are blocked by TTX (Wilson and Callaway 2000). In dopaminergic periglomerular neurons, both Ca2+ current and TTX-sensitive current are necessary for oscillatory pacemaking activity, and loss of either one produces complete quiescence in most neurons. Analysis of currents flowing between spikes
These experiments suggest that both Na+ and Ca2+ currents play important roles in driving pacemaking activity. To better quantify the ionic currents that flow during the spontaneous depolarization between spikes, we used the action-potential-clamp method. In each cell, we recorded first a 5-s segment of spontaneous activity under current clamp. Then the amplifier was switched to voltage-clamp mode, and the recorded segment was used as command waveform (Taddese and Bean 2002; Zaza et al. 1997). With this method, voltage-dependent channels are exposed to the same potential in voltage clamp as in current clamp, and ionic currents from purely voltage-dependent channels should be identical in both modes. Thus individual ionic currents flowing during and between spikes can be isolated pharmacologically or by solution exchange in the voltage-clamp experiments. Based on the current-clamp results, we focused on voltage-dependent Na+ and Ca2+ currents.
Figure 5A illustrates the brief segment of spontaneous activity recorded in current-clamp mode and administered to the same neuron as a voltage command. Figure 5B shows current carried by voltage-dependent Na+ channels in response to the command obtained by subtraction after TTX treatment (red trace). To measure Ca2+ current (blue trace), we adopted a strategy of replacing external Ca2+ ions by equimolar Co2+, which blocks current in all varieties of voltage-dependent Ca2+ channels known to be expressed in mammalian neurons. This subtraction was carried out with TEA present in both solutions to block Ca2+-activated K+ currents. As will be discussed, the replacement of Ca2+ by Co2+ could affect currents through other channels in addition to voltage-dependent Ca2+ channels, including nonvoltage-dependent Ca2+-permeant channels and Ca2+-activated nonselective cation channels. For simplicity we will refer to the current obtained by subtraction with Co2+ substitution as "Ca2+ current", while recognizing the possibility that currents carried by multiple types of Ca2+-sensitive channels may contribute to it.
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1.5–2.5 pA of inward current during the first half of the interspike interval as the voltage increased from about –60 to about –50 mV. In the later part of the interspike interval, as the voltage depolarized past –50 mV, both currents became increasingly large, with Na+ current always larger than Ca2+ current. In results collected from 11 cells, the amplitude of the Na+ current (Fig. 6A, red symbols) flowing during the interspike interval was measured at four different voltages; the average values were: –1.8 ± 1.1 pA at –60 mV, –2.9 ± 1.7 pA at –55 mV, –4.9 ± 2.8 pA at –50 mV, and –7.6 ± 3.6 pA at –45 mV. In the same 11 cells, the average values for the interspike Ca2+ current (Fig. 6A, blue symbols) were: –1.3 ± 1.2 pA at –60 mV, –1.4 ± 1.3 pA at –55 mV, –1.7 ± 1.3 pA at –50 mV, and –2.2 ± 1.7 pA at –45 mV. Thus Na+ current was on average larger in magnitude than Ca2+ current at all times during the interspike interval and also increased more steeply with voltages over the range of from –60 to –45 mV.
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Voltage dependence of Na+ and Ca2+ current
To examine the voltage-dependence of Na+ and Ca2+ currents, we delivered voltage ramps (–80 to –20 mV at 170 mVs–1). Sodium and Ca2+ currents were isolated using TTX application and Co2+ substitution, as for interspike currents. Figure 7A shows the ramp-elicited currents before and after the application of TTX under ionic conditions designed to block Ca2+ currents and reduce K+ currents (external solution with 1.8 mM CoCl2 and 5.4 mM TEA-Cl). The current blocked by TTX began to activate near –60 mV and reached a peak near –35 mV (Fig. 7C, red trace, n = 12). Figure 7B shows ramp-elicited currents in the presence of TTX before and after replacing Ca2+ by equimolar Co2+; the Ca2+ current began to activate near –50 mV and continued to increase up to –20 mV (Fig. 7C, blue trace, n = 12). Cobalt substitution always blocked all depolarization-evoked inward current remaining in TTX. In most neurons, Co2+ substitution affected ramp-evoked current only positive to about –55 mV, as in Fig. 7. In other neurons, Co2+ substitution also produced an outward shift in the current at voltages negative to –60 mV. This could represent block of an additional component of Ca2+ current that is not voltage dependent and flows at negative voltages. Alternatively, it could result from a less direct effect of eliminating Ca2+ entry, such as reduction of a Ca2+-activated nonselective cation current. Another possibility that cannot be ruled out is a change in the seal resistance between the recording pipette and the cell. Because the effect of Co2+ substitution on current negative to –60 mV was inconsistent and present only in a minority of the neurons (1/3) we did not attempt to study it further.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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found that a very similar percentage (80%) of dopaminergic periglomerular neurons studied in brain slice were spontaneously active. Their studies used a different transgenic mouse strain in which dopaminergic neurons were labeled with green fluorescent protein (GFP) driven by the tyrosine hydroxylase promoter, and the virtually identical results in the two different mouse strains argues against artifacts arising from overexpression of GFP (which is cytoplasmic) or PLAP (which is extracellular). The similarity of our results to those of Pignatelli et al. also argues against the possibility that the presence of spontaneous activity is critically dependent on the precise ionic composition of the external solution, which was somewhat different in our experiments [(in mM) 1.8 CaCl2, 1 MgCl2, 5.4 KCl, 10 glucose] than in theirs [(in mM) 2.0 CaCl2, 1 MgCl2, 2.5 KCl, 15 glucose]. When studied after dissociation, the neurons studied by Pignatelli et al. (2005) fired somewhat faster (average 14 Hz) than in our experiments (8 Hz), a difference that might be accounted for by the different ages (adult vs. P12–P17, respectively). Pignatelli et al. found that the neurons actually fired faster after dissociation (14 Hz) than in brain slice (7–8 Hz), consistent with the idea that the fundamental ionic machinery for generating pacemaking is located in the cell body and can be fruitfully studied in voltage-clamp experiments using dissociated neurons.
Unlike pacemaking in some other types of excitable cells, spontaneous activity in dopaminergic periglomerular neurons does not appear to involve Ih because neither 30 µM ZD 7288 nor 1 mM Cs+ slowed firing. In principle, Ih might play a larger role in intact cells than dissociated cells if it was present in dendrites, but as noted, firing of intact cells is slower than for dissociated cells (Pignatelli et al. 2005), perhaps due to the capacitative load of the dendritic tree. Also, Pignatelli and colleagues found that even intact dopaminergic periglomerular cells studied in brain slice do not have detectable Ih, fitting well with our results. In this respect, dopaminergic periglomerular cells appear to differ from another (still undefined) population of periglomerular cells that do have Ih (Cadetti and Belluzzi 2001). Although pacemaking activity has often been associated with Ih, this current is not required in many types of spontaneously active neurons, including cerebellar Purkinje cells (Raman and Bean 1999) and neurons of the suprachiasmatic nucleus (de Jeu and Pennartz 1997; Pennartz et al. 1997), subthalamic nucleus (Bevan and Wilson 1999; Do and Bean 2003), and tuberomammillary nucleus (Llinas and Alonso 1992; Uteshev et al. 1995). In the case of midbrain dopaminergic neurons, neurochemically and topographically distinct populations can be recognized, and Ih is actively involved in pacemaking only in a subset of them (Neuhoff et al. 2002; Seutin et al. 2001). Dopaminergic amacrine cells in the retina show pacemaking activity that does not involve Ih (Feigenspan et al. 1998), in this way resembling dopaminergic periglomerular cells of the main olfactory bulb.
A "persistent" Na+ current flowing at interspike voltages is important in driving the pacemaking activity of dopaminergic periglomerular cells. Our data suggest that this is the major current that drives pacemaking. In this respect, these neurons are similar to a number of other central neurons showing spontaneous firing, including those of the tuberomammillary (Llinas and Alonso 1992; Taddese and Bean 2002; Uteshev et al. 1995), pedunculopontine (Takakusaki and Kitai 1997), subthalamic (Bevan and Wilson 1999; Do and Bean 2003), suprachiasmatic (Jackson et al. 2004; Pennartz et al. 1997), and cerebellar nuclei (Raman et al. 2000). The properties of the persistent Na+ current we measured in acutely dissociated neurons (average magnitude 30 pA, activation midpoint approximately –50 mV) are virtually identical to those measured in intact dopaminergic periglomerular cells in brain slice (Pignatelli et al. 2005).
The spontaneous activity of dopaminergic periglomerular cells appears to differ from many other types of spontaneously active central neurons in the role of Ca2+ entry. We found that block of Ca2+ entry (by replacing Ca2+ with Co2+) consistently halted spontaneous firing of dissociated periglomerular neurons; similarly, Pignatelli and colleagues (2005) found that block of Ca2+ current (by addition of 0.1 mM Cd2+) consistently blocked spontaneous firing of periglomerular neurons studied in brain slice. In contrast, in many other types of central neurons, block of Ca2+ channels has little or no effect on spontaneous firing (e.g., Do and Bean 2003; Llinas and Alonso 1992; Pennartz et al. 1997; Raman et al. 2000; Takakusaki and Kitai 1997), whereas in others, spontaneous firing continues but with an altered pattern (Beurrier et al. 2000; Bevan and Wilson 1999). Frequently, suppression of Ca2+ current produces a net depolarization, as if the dominant electrical effect is from blocking Ca2+-activated K+ currents (Jackson et al. 2004; Pennartz et al. 2002; Raman et al. 2000). However, in dopaminergic periglomerular cells, block of Ca2+ current consistently resulted in net hyperpolarization and cessation of firing, suggesting that flow of Ca2+ current during the pacemaking cycle is not counterbalanced by an equal or greater Ca2+-activated K+ current. When measured with the action potential clamp, interspike Ca2+ current amounted to 70% of interspike Na+ current, consistent with a substantial role in pacemaking. Pignatelli and colleagues (2005) found that pacemaking was stopped by mibefradil or by 0.1 mM Ni2+, suggesting the particular importance of T-type Ca2+ channels, while blockers of L-, N-, and P-type channels did not stop firing. Interestingly, this is apparently different from dopaminergic neurons of the substantia nigra where L-type Ca2+ current appears to play a major role in pacemaking (Durante et al. 2004; Kang and Kitai 1993; Mercuri et al. 1994; Wilson and Callaway 2000).
Our experimental measurements of the ionic currents during the pacemaking cycle can be compared with the predictions of such currents made by Pignatelli et al. (2005) using a Hodgkin-Huxley-like mathematical model. Considering the small size of the currents that generate the spontaneous depolarization, and the consequent difficulty in either measuring or accurately predicting them, the experimental measurements and the model are remarkably similar. In both experiments and model, Na+ currents and Ca2+ currents are of comparable size (2–3 pA) in the mid-region of the interspike interval, with Na+ current somewhat larger and showing a greater degree of increase during the approach to spike firing. The experiments using trains of action potentials as command waveforms consistently showed a small but measurable inward current sensitive to Ca2+ replacement by Co2+ that flowed at the most negative voltages during the interspike interval, near –70 mV. This is more negative than the voltage at which clear Ca2+ current flowed in most experiments using voltage ramps (approximately –60 mV). The reason for this difference is not clear, but the current at more negative voltages can constitute a substantial part of the total current during the interspike interval that is sensitive to Ca2+ replacement by Co2+. One possibility is that this component of current during the pacemaking cycle represents "tail current" from T-type Ca2+ channels activated by the preceding spike, as predicted by the model of Pignatelli and colleagues (2005). Another interesting possibility is that some of the current at the most negative voltages represents current from the operation of the Na+/Ca2+ exchanger, which would generate steady inward current at these voltages while extruding the Ca2+ that enters during the overall pacemaking cycle (mostly during the spikes). A role for Na+/Ca2+ exchanger current in helping to drive pacemaking has been proposed in the case of cardiac pacemaking cells (Bogdanov et al. 2001; Kurata et al. 2002).
Previously, recordings from juxtaglomerular cells of undefined transmitter properties have shown a subset of cells that show spontaneous activity characterized by irregular bursting activity (Hayar et al. 2004; McQuiston and Katz 2001; Puopolo and Belluzzi 2001). This seems clearly different from the rhythmic pacemaking activity of dopaminergic periglomerular neurons and most likely represents a different cell type (or types) that remain to be identified. It is not surprising that cells characterized by regular pacemaking were not obvious in previous recordings from unlabeled cells given the small fraction (10%) of dopaminergic neurons present in the glomerular layer compared with the overall population of juxtaglomerular cells.
Pacemaking activity of dopaminergic periglomerular neurons suggests that there may be tonic release of dopamine into glomeruli at least under some circumstances. A variety of experiments show that dopamine plays a major role in modulating the sensitivity of the olfactory system to odorants. Olfactory deprivation by olfactory nerve transaction or occlusion of the nostril reduces the content of dopamine and the expression of tyrosine hydroxylase in the ipsilateral olfactory bulb by as much as 75% (Baker 1990; Baker et al. 1983). As a result, mitral cells exhibit enhanced responsiveness to odorants (Guthrie et al. 1990). A similar effect is observed on administration of the D2 antagonist spiperone (Wilson and Sullivan 1995), whereas the D2 agonist quinpirole reduces the responsiveness of mitral cells to odorants (Doty and Risser 1989).
Mechanistically, dopamine depresses synaptic transmission between olfactory receptor neurons and mitral cells by acting at presynaptic D2 receptors (Berkowicz and Trombley 2000; Coronas et al. 1997; Ennis et al. 2001; Hsia et al. 1999; Koster et al. 1999; Mansour et al. 1990), reduces the excitability of mitral cells (Nowycky et al. 1983), and interferes with the invasion of mitral cell dendrites by somatic action potentials (Davila et al. 2003; Davison et al. 2004; Duchamp-Viret et al. 1997). Overall, dopamine has an important function in setting the gain for odor discrimination, with increasing levels of dopamine raising the threshold for odorant detection and reduced levels increasing the sensitivity of the olfactory system. This is analogous to dopamine’s role in the retina where it sets the gain of the retinal networks for vision in bright light (Witkovsky and Dearry 1991). The spontaneous activity of dopaminergic periglomerular cells is well-suited for maintaining a tonic or basal concentration of dopamine in the olfactory glomeruli and outer plexiform layer. This hypothesis is supported by the observation that D2 receptor blockers reduce the spontaneous oscillations in the electrical activity of the olfactory bulb (Davison et al. 2004). In other spontaneously active dopaminergic neurons, such as those of the substantia nigra and retina, dopamine release is influenced by the firing rate of the cell and can be modulated by neurotransmitters that increase or decrease the frequency of spontaneous firing (Jaffe et al. 1998; Puopolo et al. 2001). A similar control of the firing rate of dopaminergic neurons in the olfactory bulb represents a plausible mechanism for up or down regulation of the sensitivity of the neural networks responsible for odor detection and discrimination.
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Address for reprint requests and other correspondence: E. Raviola,Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115 (E-mail: elio_raviola{at}hms.harvard.edu)
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REFERENCES |
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