Carbachol induces burst firing of dopamine cells in the ventral tegmental area by promoting calcium entry through L-type channels in the rat
1 Division of Basic Medical Sciences
2 Discipline of Psychiatry, Faculty of Medicine, Memorial University of Newfoundland, St John's NL A1B 3V6, Canada
Abstract
Enhanced activity of the central dopamine system has been implicated in many psychiatric disorders including schizophrenia and addiction. Besides terminal mechanisms that boost dopamine levels at the synapse, the cell body of dopamine cells enhances terminal dopamine concentration through encoding action potentials in bursts. This paper presents evidence that burst firing of dopamine cells in the ventral tegmental area was under cholinergic control using nystatin-perforated patch clamp recording from slice preparations. The non-selective cholinergic agonist carbachol excited the majority of recorded neurones, an action that was not affected by blocking glutamate and GABA ionotropic receptors. Twenty per cent of dopamine cells responded to carbachol with robust bursting, an effect mediated by both muscarinic and nicotinic cholinoceptors postsynaptically. Burst firing induced as such was completely dependent on calcium entry as it could be blocked by cadmium and more specifically the L-type blocker nifedipine. In the presence of the sodium channel blocker tetrodotoxin, carbachol induced membrane potential oscillation that had similar kinetics and frequency as burst firing cycles and could also be blocked by cadmium and nifedipine. Direct activation of the L-type channel with Bay K8644 induced strong bursting which could be blocked by nifedipine but not by depleting internal calcium stores. These results indicate that carbachol increases calcium entry into the postsynaptic cell through L-type channels to generate calcium-dependent membrane potential oscillation and burst firing. This could establish the L-type channel as a target for modulating the function of the central dopamine system in disease conditions.
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Introduction
The midbrain dopaminergic (DAergic) system has been implicated in a variety of diseases including schizophrenia and substance abuse as a result of enhanced DA transmission (Kiyatkin, 1995; Knable & Weinberger, 1997; Koob, 2000; Schultz, 2002). The cholinergic agonist carbachol is readily self-administered in the ventral tegmental area (VTA) (Ikemoto & Wise, 2002) and the reinforcing properties of nicotine are also mediated by cholinoceptors in the VTA (Corrigall et al. 1994; Pidoplichko et al. 2004), a brain region rich in DAergic neurones projecting to nucleus accumbens and prefrontal cortex that are involved in addiction and psychosis. DA levels can be elevated due to actions at the terminal or the cell body. At the terminal site, because released DA is taken back up to the terminal by reuptake transporters, agents such as cocaine and amphetamines that block this action effectively enhance synaptic DA levels. At the somatic site, firing in bursts has been found to be more effective in increasing DA levels (Gonon, 1988; Suaud-Chagny et al. 1992; Garris et al. 1994; Floresco et al. 2003) due to saturation of the reuptake mechanism and reduced autoreceptor inhibition (Chergui et al. 1994).
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Cholinergic activation of DA cells is well documented. Cholinergic agonists are reported to increase burst firing percentage in intact animals (Gronier & Rasmussen, 1998). However, in slice preparations, activation of nicotinic acetylcholine receptors (nAChRs) or muscarinic acetylcholine receptors (mAChRs) only increases the firing rate of DA cells (Lacey et al. 1990; Pidoplichko et al. 1997; Yin & French, 2000; Grillner & Mercuri, 2002). This inconsistency may be due to cholinergic modulation of ongoing synaptic signals in vivo or the conflicting roles of the many subtypes of cholinoceptors in regulating the firing behaviour of DA cells. For example, presynaptic nAChRs promote glutamate release and induce long-term potentiation at the glutamatergic synapses in the VTA (Mansvelder & McGehee, 2000; Mansvelder et al. 2002) as well as increase GABA release (Mansvelder et al. 2002). Similarly, mAChRs depress both excitatory and inhibitory synaptic transmission to DA cells (Grillner et al. 1999, 2000; Zheng & Johnson, 2003) and mediate a slow inhibitory synaptic potential (Fiorillo & Williams, 2000) while exciting DA cells postsynaptically (Lacey et al. 1990). Moreover, how DA cells are excited by different sources of synaptic input is also important in encoding DA cell firing patterns. Recently, it has been reported that GABAergic disinhibition increases the number of spiking cells in the VTA, whereas direct stimulation of a midbrain cholinergic cell group that innervates the VTA does not recruit more cells into firing but rather enhances burst firing of cells that are already active (Floresco et al. 2003). As the study was done in intact animals and active DA cells were detected by sampling the entire VTA with representative electrode passes, the increased percentage of burst firing following cholinergic activation may result from the assumed conversion of non-burst firing into bursting. We therefore conducted experiments in an in vitro slice preparation to directly test whether cholinergic activation converts tonically firing DA cells to burst firing and whether cholinergic activation of DA cells is dependent on actions at presynaptic elements. The hypothesis is that carbachol induces burst firing of DA cells in the VTA providing a cellular basis for the observation that carbachol is self-administered into the VTA (Ikemoto & Wise, 2002).
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Cholinergic activation mobilizes Ca2+ through a number of routes: nAChRs are permeable to Ca2+; nAChRs increase glutamate release resulting in Ca2+ entry through the NMDA receptors; mAChRs mobilize Ca2+ from internal stores; and the depolarization produced by cholinergic activation opens voltage-gated Ca2+ channels. Ca2+ has been found to play conflicting roles in burst firing. It is conventionally thought that Ca2+ entry during action potential makes the cell more excitable, thus speeding up action potential firing and further Ca2+ entry. When intracellular Ca2+ levels reach a point where Ca2+-dependent K+ channels are activated, bursting is terminated. However, it has been found that Ca2+ entry through the T-type channels is coupled to Ca2+-dependent K+ channels in DA cells and their blockade leads to a reduction in afterhyperpolarizing potential and burst firing (Wolfart & Roeper, 2002). Burst firing of DA cells induced by NMDA and apamin has been reported to persist in Ca2+-free media and following intracellular Ca2+ chelation (Johnson et al. 1992) or on the other end of the spectrum to depend entirely on Ca2+ entry into the cell (Grace & Bunney, 1984; Mercuri et al. 1994). It is therefore important to determine how Ca2+ entry through different routes during cholinergic activation regulates DA cell burst firing. This paper describes work conducted on VTA slices that cholinergic activation serves as a switch for DA cell burst firing involving primarily Ca2+ entry through the L-type Ca2+ channels on the postsynaptic cell.
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Methods
All experiments in this paper were carried out on rat brain slices using nystatin-perforated patch clamp recording. Procedures involving animal handling and tissue harvesting were in accordance with guidelines set by the Institutional Animal Care Committee at the Memorial University of Newfoundland. Sprague-Dawley rat pups of either sex, aged 5–8 days were obtained from the vivarium of Memorial University of Newfoundland and were used in experiments between 8 and 20 postnatal days.
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Slice preparation
Rats were deeply anaesthetized with halothane and killed by strong compression of the chest. The skull was quickly opened to expose the brain, which was cooled in situ with ice-cold, carbogenated artificial cerebrospinal fluid (ACSF, composition (mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 18 NaHCO3, and 11 glucose, pH 7.4 when bubbled with 95% O2 and 5% CO2). The brain was removed and placed in chilled and gassed ACSF for 30 s. A block cut from the brain containing the midbrain section was glued to a slicing stage with the base of the brain facing up. The stage was orientated in the slicing chamber, filled with cold and gassed ACSF, in such a way that cutting started from the caudal end of the block. Two 400 μm horizontal slices containing the VTA were cut on a Leica vibratome (VT1000, Heidelberger, Germany) and were allowed to recover at room temperature (22°C) for at least 1 h prior to recording. Then a slice was further trimmed to fit into a recording chamber of about 500 μl where it was submerged and continuously perfused with carbogenated ACSF at a rate of 2–3 ml min–1 at room temperature.
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Perforated patch clamp recording
Recordings were obtained using the nystatin-perforated patch technique to avoid dilution of diffusible messenger molecules that can occur during conventional whole-cell recording. The recordings were made within the confines of the VTA under a dissecting microscope (Leica MZ6). In fresh slices, the VTA is a darker area between the midline and substantia nigra, which is semi-transparent and oval in shape. In a group of separate animals, horizontal sections for this age group were stained for tyrosine hydroxylase, which revealed that DA cells were more readily found in the caudal portion of the VTA. Patch electrodes were prepared from KG-33 glass micropipettes (OD 1.5 mm, Garner Glass Co., Claremont, CA, USA) on a P-97 Brown-Flaming micropipette puller (Sutter Instruments, Novato, CA, USA). The tip of the electrode was filled with intracellular solution (composition (mM): 120 potassium acetate, 40 Hepes, 5 MgCl2, and 10 EGTA, with pH adjusted to 7.35 using 0.1 N KOH) and then back-filled with the same solution containing 450 μg ml–1 nystatin and Pluronic F127, yielding an electrode resistance of 4–6 M. Gigaohm seals were made using a Warner PC-505B (Warner Instruments Inc., Hamden, CT, USA) or a MultiClamp 700B (Axon Instruments, Foster City, CA, USA) amplifier. The signals were sampled at 5 kHz and digitized by DigiData 1320A using pCLAMP (versions 8 and 9) software (Axon Instruments).
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It usually took 10–20 min for complete partitions of nystatin into the membrane. In current clamp mode, access was reflected by the size of the action potential as many VTA cells were spontaneously active. After adequate access was attained, action potentials overshot 0 mV and measured at least 50 mV. Episodic protocols were used to induce hyperpolarization activated current (Ih) and derive passive characteristics of the cell such as current–voltage relationship and input resistance. Current pulses for Ih induction were of 1 s duration and the intervals between pulses were 8 s to allow complete recovery of Ih channels. Currents were adjusted to hyperpolarize the cell to around –110 mV and the rest of the steps were increments of 20 pA.
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Cells were identified based on their electrophysiological properties. In general, DA cells have lower basal firing rates, wider action potentials, stronger firing adaptation and a more prominent Ih expression than GABA neurones (Grace & Onn, 1989; Lacey et al. 1989; Johnson & North, 1992; Mercuri et al. 1995; Klink et al. 2001). DA cells also respond to DA with a characteristic hyperpolarization (Lacey et al. 1989; Johnson & North, 1992). In this paper, we adopted the expression of a prominent Ih at around –110 mV and an apparent DA-induced hyperpolarization as the main criteria for identifying DA cells. Due to the lack of immunocytochemical confirmation of the recorded cells, they were identified as DA-responsive cells in this paper.
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Components of extracellular and intracellular solutions were purchased from bulk distributors Fisher Scientific (Nepean, ON, USA) and VWR International (Missisauga, ON, USA). All other chemicals were obtained from Sigma (St Louis, MO, USA) and Tocris (Ellisville, MO, USA). Chemicals were dissolved in deionized water or DMSO as required. Aliquots of stock solutions were kept at –30°C. Prior to application, an aliquot was diluted to working concentration and applied to the bath. DA solution was made fresh daily with an equimolar concentration of the antioxidant disodium metabisulfite.
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Data analysis
Data were analysed offline with Mini Analysis (Synaptosoft Inc., Decatur, GA, USA) and pCLAMP software. Basal firing frequencies were averaged values of at least 3 min stable baseline recording. Ih was measured as the difference in voltage between instantaneous and steady-state readings. Analysis of firing behaviour was based on interspike intervals (ISIs) measured with the Mini Analysis program. Averaged as well as instantaneous firing frequencies were derived from those intervals. Burst firing was defined as two spikes or more in each bursting cycle at a frequency higher than non-bursting periods and separated by a post-burst hyperpolarization. Data were expressed as means ± S.E.M. Statistical comparisons were performed using paired (treatments on the same cell) or unpaired Student's t tests and values were considered significant when P < 0.05.
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Results
Nystatin-perforated whole-cell recordings were made on VTA cells identified as DA responsive according to criteria outlined in Methods. The average hyperpolarization following a brief application of 50 μM DA (within 1 min) was –7.22 ± 0.48 mV. Most cells (74%) were spontaneously active with regular or irregular firing at a low basal firing frequency of 0.28 ± 0.03 Hz; the remainder (26%) was quiescent during baseline recordings. Spiking DA cells had a more depolarized resting membrane potential than quiescent ones (–47.5 ± 0.55 and –51.1 ± 1.18 mV, respectively, P < 0.05, unpaired t test). Bath application of 20 μM carbachol excited the majority of the recorded cells with increased firing rates; 20% of them responded with characteristic bursting.
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Carbachol induces burst firing
Nineteen cells responded to 20 μM carbachol with a depolarization and burst firing. The average membrane depolarization induced by carbachol was 11.21 ± 0.66 mV (n = 19). Bursting usually started on the rising phase of carbachol-induced depolarization, followed by a varying period of depolarization block. Some cells resumed regular spiking following the depolarization block; some remained bursting for another 10 min or much longer before finally recovered to pre-test firing states (Fig. 1A). Within a bursting cycle, action potentials were fired with increasing frequencies and decreasing amplitudes followed by a pronounced post-burst hyperpolarization (Fig. 1B). Averaged intra-burst firing frequency induced by carbachol (1.67 ± 0.26 Hz) was about 10 times higher than basal tonic frequency (0.15 ± 0.04 Hz). Inter-burst intervals gradually increased after 10–30 min washout and cells became quiescent or spiking tonically at the same frequencies as before carbachol application (0.17 ± 0.05 Hz compared with pre-test frequency of 0.15 ± 0.04 Hz, P > 0.05, n = 19) accompanied by a complete recovery of membrane potential (–48.47 ± 0.8 mV compared with a pre-test value of –47.6 ± 1 mV, P > 0.05, n = 19). Inter-spike intervals (ISIs) were plotted to reveal their distributions in control conditions and following carbachol application. The bin size axis is on a logarithm scale to illustrate more clearly faster events after carbachol application. Baseline firing had longer intervals with a single peak frequency; carbachol caused a dramatic leftward shift of the peak and in addition gave rise to a smaller, secondary peak representing the frequency of bursting cycles (Fig. 1C).
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A, continuous current clamp recording from a representative cell showing that carbachol converted regular firing to bursting, followed by a period of depolarization block and intense bursting before complete recovery. B, traces on expanded time scales showing a regular (upper panel) and a burst (lower panel) firing pattern. Note that regular firing typically has a low frequency and each action potential is followed by a pronounced afterhyperpolarization, whereas burst firing has a cluster of action potentials fired at increasing frequencies followed by a steep post-burst hyperpolarization. C, plot of interspike intervals in bins of 0.2 s (for events less than 1 s), 2 s (for events between 1 and 10 s) or 20 s (for events between 10 and 100 s) as a percentage of the total number of events in control conditions and following carbachol application. Numbers in brackets are total number of events under each condition. The x-axis is on logarithm scale to show more clearly faster firing following carbachol treatment. Carbachol dramatically shifted the peak frequency to the left and gave rise to a secondary peak corresponding to the frequency of bursting cycles.
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Carbachol-induced bursting cells had an average resting membrane potential of –47.6 ± 1.0 mV and displayed typical tonic spiking at low frequencies (0.15 ± 0.04 Hz, n = 19). However, this averaged basal firing frequency was lower than the average firing rate of a group of 21 non-bursting cells recorded from slices taken from littermates on adjacent days (0.37 ± 0.06 Hz, n = 21, P < 0.05, unpaired t test). There was no difference in action potential half-width (time difference of the rising and falling phase of an action potential measured at half-amplitude) between cells that responded to carbachol with bursting (6.07 ± 0.27 ms, n = 19) and non-bursting (5.59 ± 0.44 ms, n = 21, P > 0.05, unpaired t test). Carbachol-induced bursting cells were scattered throughout the recording period of 8–20 postnatal days and no apparent clustering was noted.
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Carbachol induces excitation and bursting postsynaptically
We examined the synaptic mechanisms for carbachol-induced increased firing and bursting. In order to compare the responses to carbachol under different conditions, it was necessary to apply carbachol to the same cell more than once. We first established that repeated application of carbachol after complete recovery from the first dose caused comparable responses without apparent desensitization (n = 4). For synaptic blockade experiments, carbachol was first applied and the response was allowed to run its complete course; then a cocktail containing 100 μM APV, 10 μM CNQX and 100 μM picrotoxin (to block NMDA, AMPA and GABAA receptors, respectively) was applied for at least 5 min and the carbachol response was recorded in the presence of the cocktail. Carbachol (20 μM) caused a similar depolarization in the presence of the cocktail as carbachol alone (10.4 ± 0.68 mV compared with 9.4 ± 0.67 mV by carbachol alone; n = 5, P > 0.05, unpaired t test, Fig. 2A). Plotting the instantaneous firing frequencies shows that carbachol-induced increases in firing rate under control conditions and in the presence of the synaptic blocker cocktail were comparable (Fig. 2B). The basal firing rate of the cells in this group was 0.16 ± 0.1 Hz, which was increased to 0.70 ± 0.2 Hz by 20 μM carbachol. Application of the cocktail by itself did not change the basal firing frequency (0.19 ± 0.09 Hz), nor did it change the effect of carbachol (0.77 ± 0.26 Hz, n = 5, P > 0.05, paired t test, Fig. 2C). We further tested whether presynaptic elements were involved in carbachol-induced bursting. In three cells, carbachol induced burst firing in the presence of the synaptic blocker cocktail, suggesting that carbachol-induced bursting had the same site of action as carbachol-induced excitation in non-bursting DA cells (Fig. 2D). These results suggest that the presynaptic actions of carbachol did not contribute significantly to carbachol-induced excitation and bursting in slice preparations.
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A, continuous current clamp recording from a representative cell showing carbachol-induced increase in firing rates in control conditions and in the presence of a cocktail containing blockers at the GABAA, AMPA and NMDA site. B, instantaneous firing frequency of a representative cell showing that carbachol-induced excitations were comparable in control conditions and in the presence of the synaptic blocker cocktail. C, average firing frequency of a group of 5 cells showing that carbachol increased firing rates similarly in control conditions and in the presence of the synaptic blocker cocktail (*P < 0.05, paired t test versus respective controls). D, current clamp recording from a representative cell showing that carbachol induced burst firing in the presence of a cocktail that blocked glutamate and GABA ionotropic receptors. Insets: traces of basal firing and bursting following carbachol application on expanded time scales.
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Both nAChRs and mAChRs are involved
Nine cells were sequentially treated with carbachol, followed by another application of carbachol in the presence of a nicotinic or a muscarinic blocker or both. Carbachol (20 μM) applied for 60–120 s caused significant depolarization (9.5 ± 1.13 mV) and increased firing frequency (0.634 ± 0.11 Hz compared with baseline frequency of 0.164 ± 0.05 Hz, P < 0.05, n = 9). Nicotinic blockade appeared to be more effective in suppressing the initial depolarization induced by carbachol while muscarinic blockade affected the slower phase of the response so that it recovered earlier. There was a considerable overlapping between nicotinic and muscarinic responses because either antagonist reduced the peak depolarization induced by carbachol. On average, atropine blocked 45.7 ± 10% of carbachol-induced depolarization, which was similar to the portion blocked by mecamylamine (41.1 ± 7%) in the same group of cells (n = 9).
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Both receptor subtypes were similarly involved in carbachol-induced burst firing. In a group of three cells induced to burst with carbachol, 10 μM atropine shortened carbachol-induced bursting; combined application of atropine and mecamylamine (both at 10 μM) completely prevented carbachol-induced bursting (Fig. 3A). The preferential 42 agonist anatoxin A was used to test whether nicotinic activation only induced short bursting. In three cells that responded to 20 μM carbachol with burst firing, anatoxin A (10 μM) also induced robust and long-lasting burst firing (Fig. 3B). Both carbachol and anatoxin A caused similar changes in the coefficient of variance of ISIs calculated as the ratio of the S.D. and the mean of the ISIs for all events of a given condition (Fig. 3B, inset).
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A, continuous recording showing carbachol-induced bursting was shortened by the muscarinic antagonist atropine and was completely prevented by combined application of atropine and the nicotinic antagonist mecamylamine. B, the preferential 42 agonist anatoxin A induced burst firing similarly to carbachol in the same cell. Both responses were reversible upon washout. Histogram inset: coefficient of variance of interspike intervals showing that carbachol and anatoxin A increased coefficient of variance values in a similar manner.
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Carbachol-induced bursting depends on Ca2+ entry through L-type channels
Carbachol-induced bursting consisted of clusters of action potentials occurring on top of hump potentials which gave rise to membrane potential oscillations. The self-supporting nature of the oscillation suggests a sequential activation of multiple processes that are related to one another. Ca2+ is one such messenger molecule that can engage both depolarizing and repolarizing mechanisms depending on its concentration in the cell. We therefore tested the Ca2+ dependency of carbachol-induced burst firing with the non-selective Ca2+ channel blocker Cd2+. Indeed, Cd2+ (100–400 μM) applied for 3–5 min completely blocked membrane potential oscillation and bursting without any change of resting membrane potential (n = 5, Fig. 4A). Burst firing induced by the Ca2+-dependent K+ channel blocker apamin persisted in the presence of Cd2+ (Fig. 4B).
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A, the non-selective Ca2+ channel blocker Cd2+ reversibly blocked burst firing. B, burst firing induced by apamin could not be blocked by Cd2+. C, carbachol induced short bursting that was unaffected by blocking T-type channels with 100 μM Ni2+. D, carbachol-induced burst firing was completely prevented by the L-type blocker nifedipine.
Because membrane potential oscillation and bursting occurred at around –45 mV or below, a voltage range at which T-type Ca2+ channels operate, we applied nickel (100 μM) to see whether Ca2+ entered the cell through the T-type Ca2+ channel to support carbachol-induced bursting. We applied 100 μM nickel to cells induced to burst by carbachol. Comparing carbachol-induced bursting before and after nickel application, it was apparent that nickel had no affect (n = 4, Fig. 4C). The L-type channel has been implicated in DA cell firing regulation (Nedergaard et al. 1993; Mercuri et al. 1994; Johnson & Wu, 2004). In order to test its involvement in carbachol-induced burst firing, cells were first induced to burst and then the L-type Ca2+ channel blocker nifedipine (10 μM) was applied to see how L-type channel blockade affected the cells' ability to burst. In all five cells induced to burst by carbachol, blockade of the L-type channel with 10 μM nifedipine completely prevented bursting (Fig. 4D). In the presence of nifedipine, carbachol only increased firing frequency or caused a depolarization without firing any action potentials.
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Carbachol induces Ca2+-dependent membrane oscillation to support burst firing
The Ca2+ dependency of carbachol-induced burst firing in Fig. 4A clearly shows that burst firing and the associated membrane potential oscillation were simultaneously affected by Ca2+ channel blockade, as evidenced by the gradual recovery of the size of hump potentials along with the number of action potentials riding on them. In cells that were induced to burst by carbachol, adding 1 μM TTX revealed membrane oscillations that had similar time course and frequency as bursting cycles (n = 5, Fig. 5A). Under these conditions, subsequent addition of Cd2+ eliminated oscillation and reapplication of carbachol in the presence of Cd2+ only caused a depolarization (n = 5, Fig. 5B). Ca2+ entry through voltage-gated channels could be secondary to Na+ spikes. In order to show that Ca2+ entry initiated membrane oscillation and hence burst firing, we blocked Na+ spiking with 1 μM TTX and examined the effects of carbachol on the underlying oscillations. In four cells that were spiking regularly, bath application of 1 μM TTX eliminated spiking in a few minutes revealing a stable recording of resting membrane potential. Carbachol induced a large membrane oscillation that had similar kinetics and frequency to carbachol-induced bursting cycles. The oscillation was a series of Ca2+ waves mediated primarily by Ca2+ entry through L-type Ca2+ channels as it could be completely blocked by Cd2+ and nifedipine (Fig. 5C). These experiments indicate that burst firing induced by carbachol was dependent on Ca2+ entry through voltage-gated L-type Ca2+ channels. Ca2+ waves were not secondary to action potentials, but rather they initiated burst firing.
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A, carbachol-induced burst firing had a comparable frequency to carbachol-induced membrane oscillation in the presence of TTX in the same cell. B, in the presence of TTX, bursting was reduced to membrane oscillation that was carried by Ca2+ and could be blocked by Cd2+. Carbachol only induced a depolarization without membrane oscillation when voltage-gated Ca2+ channels were blocked. C, during basal firing, TTX prevented action potential firing and application of carbachol induced membrane oscillation. Pretreatment with the L-type Ca2+ blocker nifedipine abolished carbachol-induced membrane oscillation.
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L-type channel activator induces burst firing
If the L-type Ca2+ channel is a deciding factor for carbachol-induced bursting, it would be reasonable to expect that direct activation of the channel would induce similar firing mode switching. In eight cells that were tested with the allosteric L-type channel opener (–)-Bay K8644 (5 μM), five cells were converted to strong burst firing (Fig. 6A), two were burst-like with bursts interspersed by long periods of irregular firing, and one was without effect. The control compound (+)-Bay K8644 which also possesses antagonistic actions on the L-type channel suppressed firing of DA cells at 5 μM (n = 4) and none of the tested cells were converted to bursting (Fig. 6B). In some cells on which both analogues were tested, the active analogue was able to induce bursting following the control compound. Application of nifedipine (10 μM) either converted bursting back to regular spiking or inhibited firing altogether (Fig. 6, n = 4).
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A, continuous recording showing a regularly spiking cell responded to the L-type channel opener (–)-Bay K8644 with robust bursting. Nifedipine converted bursting back to regular spiking. B, the control analogue (+)-Bay K8644, which possesses antagonistic actions on the L-type channels, inhibited firing. Depletion of internal Ca2+ stores by pre-treatment with cyclopiazonic acid did not affect (–)-Bay K8644-induced bursting, which could be blocked by nifedipine.
, http://www.100md.com Ca2+ releasing channels on internal Ca2+ stores are regulated by intracellular Ca2+ levels and in the case of ryanodine receptors, are gated directly by the L-type channel (Berridge et al. 2000; Ouardouz et al. 2003; Power & Sah, 2005). We examined whether L-type channel activation induced similar bursting following depletion of internal Ca2+ stores. In two cells that were treated with cyclopiazonic acid (30 μM) for more than 30 min, (–)-Bay K8644 (5 μM) was still able to induce strong bursting (Fig. 6B), indicating internal Ca2+ stores are not necessary for L-type channel-mediated bursting.
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Discussion
In this paper, we observed that activation of cholinoceptors excited most cells in the VTA and induced burst firing in 20% of DA cells mediated by both muscarinic and nicotinic cholinoceptors postsynaptically. Carbachol-induced burst firing was dependent on Ca2+ entry through the L-type, but not the T-type, Ca2+ channel. Ca2+ entry through L-type channels caused membrane potential oscillation that supported the periodicity of burst firing. Direct activation of L-type channels induced similar bursting without apparent participation of internal Ca2+ stores. Our observations are significant in that cholinergic input to DA cells serves as a trigger to switch firing mode of DA cells from tonic to bursting which results from an L-type Ca2+-dependent membrane potential oscillation of the postsynaptic cell.
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Technical considerations
In our experiments, we found that a subpopulation of DA cells in VTA slices harvested from pre-weaning rats (8–20 days old) displayed burst firing patterns following cholinergic activation and direct L-type channel opening. Pacemaker-like firing at low frequencies is often seen as a predominant feature of DA cells in slice preparations. This is because DA cells in slices are deprived of synaptic input which contributes to firing irregularity and bursting (Kitai et al. 1999; Grace, 2000; Cooper, 2002; Grillner & Mercuri, 2002). Burst firing in slice preparations is rare. The robust bursting reported in this paper may have been facilitated by the perforated patch configuration which preserves the integrity of the cell allowing a more complete expression of Ca2+ and second messenger signalling. However, the exclusion of burst firing under conventional whole-cell recording conditions is not complete. Using the conventional patch clamp method, it has been reported that DA cells displaying small AHPs respond to orexin A with burst firing (Korotkova et al. 2003). Another factor is that our recordings were made at room temperature (22°C). Burst firing has been reported to be qualitatively the same at room temperature and 36–37°C except that the ISIs are a little shorter at higher temperatures (Wolfart & Roeper, 2002). Whether the temperature-sensitive Ih channels on DA cells contribute to bursting remains to be tested. The possibility that burst firing could be related to the age of rats used in this paper (8–20 postnatal days) is supported by the finding that DA neurones in slices from immature rats (15–21 days old) exhibited not only pacemaker-like firing but also irregular and bursting patterns (Mereu et al. 1997). This age-related shift in firing modes is opposite in other reports that found that burst firing is less common in immature rats (Choong & Shen, 2004). While these factors might have increased the occurrence of bursting, more work is needed to establish their contributions.
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Ca2+ influx through the L-type channel mediates bursting
The most important finding in this paper is that carbachol-induced bursting was dependent on Ca2+ influx through the L-type channels and direct activation of these channels induced similar bursting. Ca2+ has been found to play conflicting roles in DA cell bursting. Manipulation of intracellular Ca2+ levels through the recording pipette shows that increased Ca2+ underlies burst firing, an effect that can be blocked by Ca2+ chelation (Grace & Bunney, 1984) although there are opposite observations that NMDA-induced bursting persists in Ca2+-free solutions (Johnson et al. 1992). In line with this, DA cells are found to have membrane oscillation that is carried by Ca2+ channels (Grace & Onn, 1989; Kang & Kitai, 1993; Nedergaard et al. 1993; Mercuri et al. 1994). However, Ca2+ entry also promotes the activities of Ca2+-dependent K+ channels, which is a limiting factor in repetitive firing of DA cells. Consequently, blocking these channels with apamin induces strong burst firing in DA cells (Seutin et al. 1993; Ping & Shepard, 1996; Shepard & Stump, 1999). There is evidence that apamin-sensitive channels may be directly coupled to T-type Ca2+ channels; thus Ca2+ entry through T-type channels mediates firing inhibition (Wolfart & Roeper, 2002). We have observed bursting induced by carbachol or apamin under the same conditions. The clear difference is that carbachol-induced bursting was dependent on Ca2+ entry through voltage-gated Ca2+ channels whereas apamin induced bursting that persisted during Ca2+ channel blockade. The route of Ca2+ entry is therefore critical for downstream Ca2+-dependent effectors that determine the different types of burst firing in DA cells. The T-type channels are the prime target, not only because they operate at around the resting membrane potential and contribute firing irregularity (Cui et al. 2004), but also because they can be modulated by neuroleptics (Santi et al. 2002). Since psychosis has been proposed as a disease of enhanced DA transmission, if T-type channels mediate burst firing to promote DA release from the terminals, it makes perfect sense that neuroleptics curb DA transmission by reducing the T-type channel-mediated burst firing. We did not, however, observe any effects of T-type Ca2+ channel blockade on carbachol-induced bursting, nor did we observe the reported burst-promoting effects of blocking T-type channels (Wolfart & Roeper, 2002). We have shown evidence that the L-type channel was implicated in bursting because of its sensitivity to nifedipine, and direct opening of L-type channels promoted bursting in the same manner. This is consistent with findings that L-type Ca2+ channels participate in shaping up DA firing characteristics (Nedergaard et al. 1993; Mercuri et al. 1994); however, our results do not support the involvement of these channels in apamin-induced burst activities reported previously (Shepard & Stump, 1999; Johnson & Wu, 2004).
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Mechanisms of L-type channel-mediated bursting
We observed that blocking L-type Ca2+ channels abolished membrane potential oscillation and the accompanying burst firing induced by carbachol. The activation of L-type Ca2+ channels is not secondary to sodium spikes as evidenced by Ca2+ oscillation in the presence of TTX when the cells were already bursting and the sensitivity of the membrane potential oscillation to Cd2+ and nifedipine. More importantly, TTX application did not reveal Ca2+ oscillation when the cells were not bursting. Subsequent application of carbachol induced membrane oscillation, strongly indicating that they were supported by L-type channel activity to initiate carbachol-induced burst firing. One possibility that carbachol generates large Ca2+ waves is that Ca2+ influx through L-type channels induces further Ca2+ release from the internal Ca2+ stores. The Ca2+ releasing channels on the internal stores have different sensitivity to intracellular Ca2+ levels and can be directly gated by the L-type channel; the cooperation between Ca2+ influx and the internal stores may contribute to the Ca2+ oscillation we observed. We have shown that L-type channel opening induced strong bursting that persisted following depletion of internal Ca2+ stores, arguing against a significant role for Ca2+-induced Ca2+ release in burst firing. An alternative mechanism could be that Ca2+ entry through L-type channels is sustained as the result of L-type channel phosphorylation by Ca2+-dependent protein kinases to support burst firing. The resulting increase in intracellular Ca2+ could activate Ca2+-sensitive protein kinases such as protein kinase C and calmodulin kinase II, both of which are capable of phosphorylating the L-type channels making them easier to open for longer period of time (Dzhura et al. 2000; Young & Yang, 2004; Yang et al. 2005).
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L-type channels have been found to mediate a slow excitatory synaptic drive to DA cells (Bonci et al. 1998). Their involvement in bursting could be via enhancing excitatory synaptic strength. This becomes a more important issue in the case of carbachol-induced bursting since there are known synaptic mechanisms involved in cholinergic excitation of DA cells (Mansvelder & McGehee, 2000; Mansvelder et al. 2002). However, synaptic blockade at the AMPA, NMDA and GABAA site did not alter carbachol-induced bursting, suggesting that in slices presynaptic elements do not carry active signals for cholinoceptors to modulate or different receptor subtypes to negate each others action at the terminal. Postsynaptically, activation of L-type channels may release DA from dendrites, which in turn terminates firing by D2-mediated autoinhibition. This autoinhibition may come in cycles similar to bursting because of somatodendritic DA reuptake mechanism. It has been shown that somatodendritic DA release is Ca2+ dependent and may contribute to DA cell bursting (Chen & Rice, 2001; Beckstead et al. 2004). It would be interesting to test whether this is the case for carbachol-induced bursting.
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How L-type channels operate at near-resting membrane potential to mediate burst firing may be explained by channel diversity and phosphorylation. There are reports that some L-type Ca2+ channels (Cav1.3, Cav1.4) are active at much more negative potentials than conventional high voltage-gated Ca2+ channels (Durante et al. 2004; Lipscombe et al. 2004; Power & Sah, 2005). In fact, small depolarizations preferentially open L-type channels on midbrain DA neurones (Durante et al. 2004), suggesting a more significant role for L-type channels in firing regulation. Alternatively, L-type channels may undergo Ca2+-dependent phosphorylation to facilitate opening (Dzhura et al. 2000).
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In conclusion, activation of cholinoceptors in the VTA leads to direct opening of L-type Ca2+ channels on the postsynaptic cell that gives rise to membrane oscillation and bursting. The involvement of L-type channels in DA cell bursting may represent a promising target for modulating the central DA system in disease conditions as supported by a finding that L-type blockers help relieve craving in drug addicts (Shulman et al. 1998).
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2 Discipline of Psychiatry, Faculty of Medicine, Memorial University of Newfoundland, St John's NL A1B 3V6, Canada
Abstract
Enhanced activity of the central dopamine system has been implicated in many psychiatric disorders including schizophrenia and addiction. Besides terminal mechanisms that boost dopamine levels at the synapse, the cell body of dopamine cells enhances terminal dopamine concentration through encoding action potentials in bursts. This paper presents evidence that burst firing of dopamine cells in the ventral tegmental area was under cholinergic control using nystatin-perforated patch clamp recording from slice preparations. The non-selective cholinergic agonist carbachol excited the majority of recorded neurones, an action that was not affected by blocking glutamate and GABA ionotropic receptors. Twenty per cent of dopamine cells responded to carbachol with robust bursting, an effect mediated by both muscarinic and nicotinic cholinoceptors postsynaptically. Burst firing induced as such was completely dependent on calcium entry as it could be blocked by cadmium and more specifically the L-type blocker nifedipine. In the presence of the sodium channel blocker tetrodotoxin, carbachol induced membrane potential oscillation that had similar kinetics and frequency as burst firing cycles and could also be blocked by cadmium and nifedipine. Direct activation of the L-type channel with Bay K8644 induced strong bursting which could be blocked by nifedipine but not by depleting internal calcium stores. These results indicate that carbachol increases calcium entry into the postsynaptic cell through L-type channels to generate calcium-dependent membrane potential oscillation and burst firing. This could establish the L-type channel as a target for modulating the function of the central dopamine system in disease conditions.
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Introduction
The midbrain dopaminergic (DAergic) system has been implicated in a variety of diseases including schizophrenia and substance abuse as a result of enhanced DA transmission (Kiyatkin, 1995; Knable & Weinberger, 1997; Koob, 2000; Schultz, 2002). The cholinergic agonist carbachol is readily self-administered in the ventral tegmental area (VTA) (Ikemoto & Wise, 2002) and the reinforcing properties of nicotine are also mediated by cholinoceptors in the VTA (Corrigall et al. 1994; Pidoplichko et al. 2004), a brain region rich in DAergic neurones projecting to nucleus accumbens and prefrontal cortex that are involved in addiction and psychosis. DA levels can be elevated due to actions at the terminal or the cell body. At the terminal site, because released DA is taken back up to the terminal by reuptake transporters, agents such as cocaine and amphetamines that block this action effectively enhance synaptic DA levels. At the somatic site, firing in bursts has been found to be more effective in increasing DA levels (Gonon, 1988; Suaud-Chagny et al. 1992; Garris et al. 1994; Floresco et al. 2003) due to saturation of the reuptake mechanism and reduced autoreceptor inhibition (Chergui et al. 1994).
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Cholinergic activation of DA cells is well documented. Cholinergic agonists are reported to increase burst firing percentage in intact animals (Gronier & Rasmussen, 1998). However, in slice preparations, activation of nicotinic acetylcholine receptors (nAChRs) or muscarinic acetylcholine receptors (mAChRs) only increases the firing rate of DA cells (Lacey et al. 1990; Pidoplichko et al. 1997; Yin & French, 2000; Grillner & Mercuri, 2002). This inconsistency may be due to cholinergic modulation of ongoing synaptic signals in vivo or the conflicting roles of the many subtypes of cholinoceptors in regulating the firing behaviour of DA cells. For example, presynaptic nAChRs promote glutamate release and induce long-term potentiation at the glutamatergic synapses in the VTA (Mansvelder & McGehee, 2000; Mansvelder et al. 2002) as well as increase GABA release (Mansvelder et al. 2002). Similarly, mAChRs depress both excitatory and inhibitory synaptic transmission to DA cells (Grillner et al. 1999, 2000; Zheng & Johnson, 2003) and mediate a slow inhibitory synaptic potential (Fiorillo & Williams, 2000) while exciting DA cells postsynaptically (Lacey et al. 1990). Moreover, how DA cells are excited by different sources of synaptic input is also important in encoding DA cell firing patterns. Recently, it has been reported that GABAergic disinhibition increases the number of spiking cells in the VTA, whereas direct stimulation of a midbrain cholinergic cell group that innervates the VTA does not recruit more cells into firing but rather enhances burst firing of cells that are already active (Floresco et al. 2003). As the study was done in intact animals and active DA cells were detected by sampling the entire VTA with representative electrode passes, the increased percentage of burst firing following cholinergic activation may result from the assumed conversion of non-burst firing into bursting. We therefore conducted experiments in an in vitro slice preparation to directly test whether cholinergic activation converts tonically firing DA cells to burst firing and whether cholinergic activation of DA cells is dependent on actions at presynaptic elements. The hypothesis is that carbachol induces burst firing of DA cells in the VTA providing a cellular basis for the observation that carbachol is self-administered into the VTA (Ikemoto & Wise, 2002).
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Cholinergic activation mobilizes Ca2+ through a number of routes: nAChRs are permeable to Ca2+; nAChRs increase glutamate release resulting in Ca2+ entry through the NMDA receptors; mAChRs mobilize Ca2+ from internal stores; and the depolarization produced by cholinergic activation opens voltage-gated Ca2+ channels. Ca2+ has been found to play conflicting roles in burst firing. It is conventionally thought that Ca2+ entry during action potential makes the cell more excitable, thus speeding up action potential firing and further Ca2+ entry. When intracellular Ca2+ levels reach a point where Ca2+-dependent K+ channels are activated, bursting is terminated. However, it has been found that Ca2+ entry through the T-type channels is coupled to Ca2+-dependent K+ channels in DA cells and their blockade leads to a reduction in afterhyperpolarizing potential and burst firing (Wolfart & Roeper, 2002). Burst firing of DA cells induced by NMDA and apamin has been reported to persist in Ca2+-free media and following intracellular Ca2+ chelation (Johnson et al. 1992) or on the other end of the spectrum to depend entirely on Ca2+ entry into the cell (Grace & Bunney, 1984; Mercuri et al. 1994). It is therefore important to determine how Ca2+ entry through different routes during cholinergic activation regulates DA cell burst firing. This paper describes work conducted on VTA slices that cholinergic activation serves as a switch for DA cell burst firing involving primarily Ca2+ entry through the L-type Ca2+ channels on the postsynaptic cell.
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Methods
All experiments in this paper were carried out on rat brain slices using nystatin-perforated patch clamp recording. Procedures involving animal handling and tissue harvesting were in accordance with guidelines set by the Institutional Animal Care Committee at the Memorial University of Newfoundland. Sprague-Dawley rat pups of either sex, aged 5–8 days were obtained from the vivarium of Memorial University of Newfoundland and were used in experiments between 8 and 20 postnatal days.
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Slice preparation
Rats were deeply anaesthetized with halothane and killed by strong compression of the chest. The skull was quickly opened to expose the brain, which was cooled in situ with ice-cold, carbogenated artificial cerebrospinal fluid (ACSF, composition (mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 18 NaHCO3, and 11 glucose, pH 7.4 when bubbled with 95% O2 and 5% CO2). The brain was removed and placed in chilled and gassed ACSF for 30 s. A block cut from the brain containing the midbrain section was glued to a slicing stage with the base of the brain facing up. The stage was orientated in the slicing chamber, filled with cold and gassed ACSF, in such a way that cutting started from the caudal end of the block. Two 400 μm horizontal slices containing the VTA were cut on a Leica vibratome (VT1000, Heidelberger, Germany) and were allowed to recover at room temperature (22°C) for at least 1 h prior to recording. Then a slice was further trimmed to fit into a recording chamber of about 500 μl where it was submerged and continuously perfused with carbogenated ACSF at a rate of 2–3 ml min–1 at room temperature.
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Perforated patch clamp recording
Recordings were obtained using the nystatin-perforated patch technique to avoid dilution of diffusible messenger molecules that can occur during conventional whole-cell recording. The recordings were made within the confines of the VTA under a dissecting microscope (Leica MZ6). In fresh slices, the VTA is a darker area between the midline and substantia nigra, which is semi-transparent and oval in shape. In a group of separate animals, horizontal sections for this age group were stained for tyrosine hydroxylase, which revealed that DA cells were more readily found in the caudal portion of the VTA. Patch electrodes were prepared from KG-33 glass micropipettes (OD 1.5 mm, Garner Glass Co., Claremont, CA, USA) on a P-97 Brown-Flaming micropipette puller (Sutter Instruments, Novato, CA, USA). The tip of the electrode was filled with intracellular solution (composition (mM): 120 potassium acetate, 40 Hepes, 5 MgCl2, and 10 EGTA, with pH adjusted to 7.35 using 0.1 N KOH) and then back-filled with the same solution containing 450 μg ml–1 nystatin and Pluronic F127, yielding an electrode resistance of 4–6 M. Gigaohm seals were made using a Warner PC-505B (Warner Instruments Inc., Hamden, CT, USA) or a MultiClamp 700B (Axon Instruments, Foster City, CA, USA) amplifier. The signals were sampled at 5 kHz and digitized by DigiData 1320A using pCLAMP (versions 8 and 9) software (Axon Instruments).
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It usually took 10–20 min for complete partitions of nystatin into the membrane. In current clamp mode, access was reflected by the size of the action potential as many VTA cells were spontaneously active. After adequate access was attained, action potentials overshot 0 mV and measured at least 50 mV. Episodic protocols were used to induce hyperpolarization activated current (Ih) and derive passive characteristics of the cell such as current–voltage relationship and input resistance. Current pulses for Ih induction were of 1 s duration and the intervals between pulses were 8 s to allow complete recovery of Ih channels. Currents were adjusted to hyperpolarize the cell to around –110 mV and the rest of the steps were increments of 20 pA.
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Cells were identified based on their electrophysiological properties. In general, DA cells have lower basal firing rates, wider action potentials, stronger firing adaptation and a more prominent Ih expression than GABA neurones (Grace & Onn, 1989; Lacey et al. 1989; Johnson & North, 1992; Mercuri et al. 1995; Klink et al. 2001). DA cells also respond to DA with a characteristic hyperpolarization (Lacey et al. 1989; Johnson & North, 1992). In this paper, we adopted the expression of a prominent Ih at around –110 mV and an apparent DA-induced hyperpolarization as the main criteria for identifying DA cells. Due to the lack of immunocytochemical confirmation of the recorded cells, they were identified as DA-responsive cells in this paper.
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Components of extracellular and intracellular solutions were purchased from bulk distributors Fisher Scientific (Nepean, ON, USA) and VWR International (Missisauga, ON, USA). All other chemicals were obtained from Sigma (St Louis, MO, USA) and Tocris (Ellisville, MO, USA). Chemicals were dissolved in deionized water or DMSO as required. Aliquots of stock solutions were kept at –30°C. Prior to application, an aliquot was diluted to working concentration and applied to the bath. DA solution was made fresh daily with an equimolar concentration of the antioxidant disodium metabisulfite.
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Data analysis
Data were analysed offline with Mini Analysis (Synaptosoft Inc., Decatur, GA, USA) and pCLAMP software. Basal firing frequencies were averaged values of at least 3 min stable baseline recording. Ih was measured as the difference in voltage between instantaneous and steady-state readings. Analysis of firing behaviour was based on interspike intervals (ISIs) measured with the Mini Analysis program. Averaged as well as instantaneous firing frequencies were derived from those intervals. Burst firing was defined as two spikes or more in each bursting cycle at a frequency higher than non-bursting periods and separated by a post-burst hyperpolarization. Data were expressed as means ± S.E.M. Statistical comparisons were performed using paired (treatments on the same cell) or unpaired Student's t tests and values were considered significant when P < 0.05.
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Results
Nystatin-perforated whole-cell recordings were made on VTA cells identified as DA responsive according to criteria outlined in Methods. The average hyperpolarization following a brief application of 50 μM DA (within 1 min) was –7.22 ± 0.48 mV. Most cells (74%) were spontaneously active with regular or irregular firing at a low basal firing frequency of 0.28 ± 0.03 Hz; the remainder (26%) was quiescent during baseline recordings. Spiking DA cells had a more depolarized resting membrane potential than quiescent ones (–47.5 ± 0.55 and –51.1 ± 1.18 mV, respectively, P < 0.05, unpaired t test). Bath application of 20 μM carbachol excited the majority of the recorded cells with increased firing rates; 20% of them responded with characteristic bursting.
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Carbachol induces burst firing
Nineteen cells responded to 20 μM carbachol with a depolarization and burst firing. The average membrane depolarization induced by carbachol was 11.21 ± 0.66 mV (n = 19). Bursting usually started on the rising phase of carbachol-induced depolarization, followed by a varying period of depolarization block. Some cells resumed regular spiking following the depolarization block; some remained bursting for another 10 min or much longer before finally recovered to pre-test firing states (Fig. 1A). Within a bursting cycle, action potentials were fired with increasing frequencies and decreasing amplitudes followed by a pronounced post-burst hyperpolarization (Fig. 1B). Averaged intra-burst firing frequency induced by carbachol (1.67 ± 0.26 Hz) was about 10 times higher than basal tonic frequency (0.15 ± 0.04 Hz). Inter-burst intervals gradually increased after 10–30 min washout and cells became quiescent or spiking tonically at the same frequencies as before carbachol application (0.17 ± 0.05 Hz compared with pre-test frequency of 0.15 ± 0.04 Hz, P > 0.05, n = 19) accompanied by a complete recovery of membrane potential (–48.47 ± 0.8 mV compared with a pre-test value of –47.6 ± 1 mV, P > 0.05, n = 19). Inter-spike intervals (ISIs) were plotted to reveal their distributions in control conditions and following carbachol application. The bin size axis is on a logarithm scale to illustrate more clearly faster events after carbachol application. Baseline firing had longer intervals with a single peak frequency; carbachol caused a dramatic leftward shift of the peak and in addition gave rise to a smaller, secondary peak representing the frequency of bursting cycles (Fig. 1C).
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A, continuous current clamp recording from a representative cell showing that carbachol converted regular firing to bursting, followed by a period of depolarization block and intense bursting before complete recovery. B, traces on expanded time scales showing a regular (upper panel) and a burst (lower panel) firing pattern. Note that regular firing typically has a low frequency and each action potential is followed by a pronounced afterhyperpolarization, whereas burst firing has a cluster of action potentials fired at increasing frequencies followed by a steep post-burst hyperpolarization. C, plot of interspike intervals in bins of 0.2 s (for events less than 1 s), 2 s (for events between 1 and 10 s) or 20 s (for events between 10 and 100 s) as a percentage of the total number of events in control conditions and following carbachol application. Numbers in brackets are total number of events under each condition. The x-axis is on logarithm scale to show more clearly faster firing following carbachol treatment. Carbachol dramatically shifted the peak frequency to the left and gave rise to a secondary peak corresponding to the frequency of bursting cycles.
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Carbachol-induced bursting cells had an average resting membrane potential of –47.6 ± 1.0 mV and displayed typical tonic spiking at low frequencies (0.15 ± 0.04 Hz, n = 19). However, this averaged basal firing frequency was lower than the average firing rate of a group of 21 non-bursting cells recorded from slices taken from littermates on adjacent days (0.37 ± 0.06 Hz, n = 21, P < 0.05, unpaired t test). There was no difference in action potential half-width (time difference of the rising and falling phase of an action potential measured at half-amplitude) between cells that responded to carbachol with bursting (6.07 ± 0.27 ms, n = 19) and non-bursting (5.59 ± 0.44 ms, n = 21, P > 0.05, unpaired t test). Carbachol-induced bursting cells were scattered throughout the recording period of 8–20 postnatal days and no apparent clustering was noted.
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Carbachol induces excitation and bursting postsynaptically
We examined the synaptic mechanisms for carbachol-induced increased firing and bursting. In order to compare the responses to carbachol under different conditions, it was necessary to apply carbachol to the same cell more than once. We first established that repeated application of carbachol after complete recovery from the first dose caused comparable responses without apparent desensitization (n = 4). For synaptic blockade experiments, carbachol was first applied and the response was allowed to run its complete course; then a cocktail containing 100 μM APV, 10 μM CNQX and 100 μM picrotoxin (to block NMDA, AMPA and GABAA receptors, respectively) was applied for at least 5 min and the carbachol response was recorded in the presence of the cocktail. Carbachol (20 μM) caused a similar depolarization in the presence of the cocktail as carbachol alone (10.4 ± 0.68 mV compared with 9.4 ± 0.67 mV by carbachol alone; n = 5, P > 0.05, unpaired t test, Fig. 2A). Plotting the instantaneous firing frequencies shows that carbachol-induced increases in firing rate under control conditions and in the presence of the synaptic blocker cocktail were comparable (Fig. 2B). The basal firing rate of the cells in this group was 0.16 ± 0.1 Hz, which was increased to 0.70 ± 0.2 Hz by 20 μM carbachol. Application of the cocktail by itself did not change the basal firing frequency (0.19 ± 0.09 Hz), nor did it change the effect of carbachol (0.77 ± 0.26 Hz, n = 5, P > 0.05, paired t test, Fig. 2C). We further tested whether presynaptic elements were involved in carbachol-induced bursting. In three cells, carbachol induced burst firing in the presence of the synaptic blocker cocktail, suggesting that carbachol-induced bursting had the same site of action as carbachol-induced excitation in non-bursting DA cells (Fig. 2D). These results suggest that the presynaptic actions of carbachol did not contribute significantly to carbachol-induced excitation and bursting in slice preparations.
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A, continuous current clamp recording from a representative cell showing carbachol-induced increase in firing rates in control conditions and in the presence of a cocktail containing blockers at the GABAA, AMPA and NMDA site. B, instantaneous firing frequency of a representative cell showing that carbachol-induced excitations were comparable in control conditions and in the presence of the synaptic blocker cocktail. C, average firing frequency of a group of 5 cells showing that carbachol increased firing rates similarly in control conditions and in the presence of the synaptic blocker cocktail (*P < 0.05, paired t test versus respective controls). D, current clamp recording from a representative cell showing that carbachol induced burst firing in the presence of a cocktail that blocked glutamate and GABA ionotropic receptors. Insets: traces of basal firing and bursting following carbachol application on expanded time scales.
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Both nAChRs and mAChRs are involved
Nine cells were sequentially treated with carbachol, followed by another application of carbachol in the presence of a nicotinic or a muscarinic blocker or both. Carbachol (20 μM) applied for 60–120 s caused significant depolarization (9.5 ± 1.13 mV) and increased firing frequency (0.634 ± 0.11 Hz compared with baseline frequency of 0.164 ± 0.05 Hz, P < 0.05, n = 9). Nicotinic blockade appeared to be more effective in suppressing the initial depolarization induced by carbachol while muscarinic blockade affected the slower phase of the response so that it recovered earlier. There was a considerable overlapping between nicotinic and muscarinic responses because either antagonist reduced the peak depolarization induced by carbachol. On average, atropine blocked 45.7 ± 10% of carbachol-induced depolarization, which was similar to the portion blocked by mecamylamine (41.1 ± 7%) in the same group of cells (n = 9).
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Both receptor subtypes were similarly involved in carbachol-induced burst firing. In a group of three cells induced to burst with carbachol, 10 μM atropine shortened carbachol-induced bursting; combined application of atropine and mecamylamine (both at 10 μM) completely prevented carbachol-induced bursting (Fig. 3A). The preferential 42 agonist anatoxin A was used to test whether nicotinic activation only induced short bursting. In three cells that responded to 20 μM carbachol with burst firing, anatoxin A (10 μM) also induced robust and long-lasting burst firing (Fig. 3B). Both carbachol and anatoxin A caused similar changes in the coefficient of variance of ISIs calculated as the ratio of the S.D. and the mean of the ISIs for all events of a given condition (Fig. 3B, inset).
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A, continuous recording showing carbachol-induced bursting was shortened by the muscarinic antagonist atropine and was completely prevented by combined application of atropine and the nicotinic antagonist mecamylamine. B, the preferential 42 agonist anatoxin A induced burst firing similarly to carbachol in the same cell. Both responses were reversible upon washout. Histogram inset: coefficient of variance of interspike intervals showing that carbachol and anatoxin A increased coefficient of variance values in a similar manner.
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Carbachol-induced bursting depends on Ca2+ entry through L-type channels
Carbachol-induced bursting consisted of clusters of action potentials occurring on top of hump potentials which gave rise to membrane potential oscillations. The self-supporting nature of the oscillation suggests a sequential activation of multiple processes that are related to one another. Ca2+ is one such messenger molecule that can engage both depolarizing and repolarizing mechanisms depending on its concentration in the cell. We therefore tested the Ca2+ dependency of carbachol-induced burst firing with the non-selective Ca2+ channel blocker Cd2+. Indeed, Cd2+ (100–400 μM) applied for 3–5 min completely blocked membrane potential oscillation and bursting without any change of resting membrane potential (n = 5, Fig. 4A). Burst firing induced by the Ca2+-dependent K+ channel blocker apamin persisted in the presence of Cd2+ (Fig. 4B).
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A, the non-selective Ca2+ channel blocker Cd2+ reversibly blocked burst firing. B, burst firing induced by apamin could not be blocked by Cd2+. C, carbachol induced short bursting that was unaffected by blocking T-type channels with 100 μM Ni2+. D, carbachol-induced burst firing was completely prevented by the L-type blocker nifedipine.
Because membrane potential oscillation and bursting occurred at around –45 mV or below, a voltage range at which T-type Ca2+ channels operate, we applied nickel (100 μM) to see whether Ca2+ entered the cell through the T-type Ca2+ channel to support carbachol-induced bursting. We applied 100 μM nickel to cells induced to burst by carbachol. Comparing carbachol-induced bursting before and after nickel application, it was apparent that nickel had no affect (n = 4, Fig. 4C). The L-type channel has been implicated in DA cell firing regulation (Nedergaard et al. 1993; Mercuri et al. 1994; Johnson & Wu, 2004). In order to test its involvement in carbachol-induced burst firing, cells were first induced to burst and then the L-type Ca2+ channel blocker nifedipine (10 μM) was applied to see how L-type channel blockade affected the cells' ability to burst. In all five cells induced to burst by carbachol, blockade of the L-type channel with 10 μM nifedipine completely prevented bursting (Fig. 4D). In the presence of nifedipine, carbachol only increased firing frequency or caused a depolarization without firing any action potentials.
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Carbachol induces Ca2+-dependent membrane oscillation to support burst firing
The Ca2+ dependency of carbachol-induced burst firing in Fig. 4A clearly shows that burst firing and the associated membrane potential oscillation were simultaneously affected by Ca2+ channel blockade, as evidenced by the gradual recovery of the size of hump potentials along with the number of action potentials riding on them. In cells that were induced to burst by carbachol, adding 1 μM TTX revealed membrane oscillations that had similar time course and frequency as bursting cycles (n = 5, Fig. 5A). Under these conditions, subsequent addition of Cd2+ eliminated oscillation and reapplication of carbachol in the presence of Cd2+ only caused a depolarization (n = 5, Fig. 5B). Ca2+ entry through voltage-gated channels could be secondary to Na+ spikes. In order to show that Ca2+ entry initiated membrane oscillation and hence burst firing, we blocked Na+ spiking with 1 μM TTX and examined the effects of carbachol on the underlying oscillations. In four cells that were spiking regularly, bath application of 1 μM TTX eliminated spiking in a few minutes revealing a stable recording of resting membrane potential. Carbachol induced a large membrane oscillation that had similar kinetics and frequency to carbachol-induced bursting cycles. The oscillation was a series of Ca2+ waves mediated primarily by Ca2+ entry through L-type Ca2+ channels as it could be completely blocked by Cd2+ and nifedipine (Fig. 5C). These experiments indicate that burst firing induced by carbachol was dependent on Ca2+ entry through voltage-gated L-type Ca2+ channels. Ca2+ waves were not secondary to action potentials, but rather they initiated burst firing.
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A, carbachol-induced burst firing had a comparable frequency to carbachol-induced membrane oscillation in the presence of TTX in the same cell. B, in the presence of TTX, bursting was reduced to membrane oscillation that was carried by Ca2+ and could be blocked by Cd2+. Carbachol only induced a depolarization without membrane oscillation when voltage-gated Ca2+ channels were blocked. C, during basal firing, TTX prevented action potential firing and application of carbachol induced membrane oscillation. Pretreatment with the L-type Ca2+ blocker nifedipine abolished carbachol-induced membrane oscillation.
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L-type channel activator induces burst firing
If the L-type Ca2+ channel is a deciding factor for carbachol-induced bursting, it would be reasonable to expect that direct activation of the channel would induce similar firing mode switching. In eight cells that were tested with the allosteric L-type channel opener (–)-Bay K8644 (5 μM), five cells were converted to strong burst firing (Fig. 6A), two were burst-like with bursts interspersed by long periods of irregular firing, and one was without effect. The control compound (+)-Bay K8644 which also possesses antagonistic actions on the L-type channel suppressed firing of DA cells at 5 μM (n = 4) and none of the tested cells were converted to bursting (Fig. 6B). In some cells on which both analogues were tested, the active analogue was able to induce bursting following the control compound. Application of nifedipine (10 μM) either converted bursting back to regular spiking or inhibited firing altogether (Fig. 6, n = 4).
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A, continuous recording showing a regularly spiking cell responded to the L-type channel opener (–)-Bay K8644 with robust bursting. Nifedipine converted bursting back to regular spiking. B, the control analogue (+)-Bay K8644, which possesses antagonistic actions on the L-type channels, inhibited firing. Depletion of internal Ca2+ stores by pre-treatment with cyclopiazonic acid did not affect (–)-Bay K8644-induced bursting, which could be blocked by nifedipine.
, http://www.100md.com Ca2+ releasing channels on internal Ca2+ stores are regulated by intracellular Ca2+ levels and in the case of ryanodine receptors, are gated directly by the L-type channel (Berridge et al. 2000; Ouardouz et al. 2003; Power & Sah, 2005). We examined whether L-type channel activation induced similar bursting following depletion of internal Ca2+ stores. In two cells that were treated with cyclopiazonic acid (30 μM) for more than 30 min, (–)-Bay K8644 (5 μM) was still able to induce strong bursting (Fig. 6B), indicating internal Ca2+ stores are not necessary for L-type channel-mediated bursting.
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Discussion
In this paper, we observed that activation of cholinoceptors excited most cells in the VTA and induced burst firing in 20% of DA cells mediated by both muscarinic and nicotinic cholinoceptors postsynaptically. Carbachol-induced burst firing was dependent on Ca2+ entry through the L-type, but not the T-type, Ca2+ channel. Ca2+ entry through L-type channels caused membrane potential oscillation that supported the periodicity of burst firing. Direct activation of L-type channels induced similar bursting without apparent participation of internal Ca2+ stores. Our observations are significant in that cholinergic input to DA cells serves as a trigger to switch firing mode of DA cells from tonic to bursting which results from an L-type Ca2+-dependent membrane potential oscillation of the postsynaptic cell.
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Technical considerations
In our experiments, we found that a subpopulation of DA cells in VTA slices harvested from pre-weaning rats (8–20 days old) displayed burst firing patterns following cholinergic activation and direct L-type channel opening. Pacemaker-like firing at low frequencies is often seen as a predominant feature of DA cells in slice preparations. This is because DA cells in slices are deprived of synaptic input which contributes to firing irregularity and bursting (Kitai et al. 1999; Grace, 2000; Cooper, 2002; Grillner & Mercuri, 2002). Burst firing in slice preparations is rare. The robust bursting reported in this paper may have been facilitated by the perforated patch configuration which preserves the integrity of the cell allowing a more complete expression of Ca2+ and second messenger signalling. However, the exclusion of burst firing under conventional whole-cell recording conditions is not complete. Using the conventional patch clamp method, it has been reported that DA cells displaying small AHPs respond to orexin A with burst firing (Korotkova et al. 2003). Another factor is that our recordings were made at room temperature (22°C). Burst firing has been reported to be qualitatively the same at room temperature and 36–37°C except that the ISIs are a little shorter at higher temperatures (Wolfart & Roeper, 2002). Whether the temperature-sensitive Ih channels on DA cells contribute to bursting remains to be tested. The possibility that burst firing could be related to the age of rats used in this paper (8–20 postnatal days) is supported by the finding that DA neurones in slices from immature rats (15–21 days old) exhibited not only pacemaker-like firing but also irregular and bursting patterns (Mereu et al. 1997). This age-related shift in firing modes is opposite in other reports that found that burst firing is less common in immature rats (Choong & Shen, 2004). While these factors might have increased the occurrence of bursting, more work is needed to establish their contributions.
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Ca2+ influx through the L-type channel mediates bursting
The most important finding in this paper is that carbachol-induced bursting was dependent on Ca2+ influx through the L-type channels and direct activation of these channels induced similar bursting. Ca2+ has been found to play conflicting roles in DA cell bursting. Manipulation of intracellular Ca2+ levels through the recording pipette shows that increased Ca2+ underlies burst firing, an effect that can be blocked by Ca2+ chelation (Grace & Bunney, 1984) although there are opposite observations that NMDA-induced bursting persists in Ca2+-free solutions (Johnson et al. 1992). In line with this, DA cells are found to have membrane oscillation that is carried by Ca2+ channels (Grace & Onn, 1989; Kang & Kitai, 1993; Nedergaard et al. 1993; Mercuri et al. 1994). However, Ca2+ entry also promotes the activities of Ca2+-dependent K+ channels, which is a limiting factor in repetitive firing of DA cells. Consequently, blocking these channels with apamin induces strong burst firing in DA cells (Seutin et al. 1993; Ping & Shepard, 1996; Shepard & Stump, 1999). There is evidence that apamin-sensitive channels may be directly coupled to T-type Ca2+ channels; thus Ca2+ entry through T-type channels mediates firing inhibition (Wolfart & Roeper, 2002). We have observed bursting induced by carbachol or apamin under the same conditions. The clear difference is that carbachol-induced bursting was dependent on Ca2+ entry through voltage-gated Ca2+ channels whereas apamin induced bursting that persisted during Ca2+ channel blockade. The route of Ca2+ entry is therefore critical for downstream Ca2+-dependent effectors that determine the different types of burst firing in DA cells. The T-type channels are the prime target, not only because they operate at around the resting membrane potential and contribute firing irregularity (Cui et al. 2004), but also because they can be modulated by neuroleptics (Santi et al. 2002). Since psychosis has been proposed as a disease of enhanced DA transmission, if T-type channels mediate burst firing to promote DA release from the terminals, it makes perfect sense that neuroleptics curb DA transmission by reducing the T-type channel-mediated burst firing. We did not, however, observe any effects of T-type Ca2+ channel blockade on carbachol-induced bursting, nor did we observe the reported burst-promoting effects of blocking T-type channels (Wolfart & Roeper, 2002). We have shown evidence that the L-type channel was implicated in bursting because of its sensitivity to nifedipine, and direct opening of L-type channels promoted bursting in the same manner. This is consistent with findings that L-type Ca2+ channels participate in shaping up DA firing characteristics (Nedergaard et al. 1993; Mercuri et al. 1994); however, our results do not support the involvement of these channels in apamin-induced burst activities reported previously (Shepard & Stump, 1999; Johnson & Wu, 2004).
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Mechanisms of L-type channel-mediated bursting
We observed that blocking L-type Ca2+ channels abolished membrane potential oscillation and the accompanying burst firing induced by carbachol. The activation of L-type Ca2+ channels is not secondary to sodium spikes as evidenced by Ca2+ oscillation in the presence of TTX when the cells were already bursting and the sensitivity of the membrane potential oscillation to Cd2+ and nifedipine. More importantly, TTX application did not reveal Ca2+ oscillation when the cells were not bursting. Subsequent application of carbachol induced membrane oscillation, strongly indicating that they were supported by L-type channel activity to initiate carbachol-induced burst firing. One possibility that carbachol generates large Ca2+ waves is that Ca2+ influx through L-type channels induces further Ca2+ release from the internal Ca2+ stores. The Ca2+ releasing channels on the internal stores have different sensitivity to intracellular Ca2+ levels and can be directly gated by the L-type channel; the cooperation between Ca2+ influx and the internal stores may contribute to the Ca2+ oscillation we observed. We have shown that L-type channel opening induced strong bursting that persisted following depletion of internal Ca2+ stores, arguing against a significant role for Ca2+-induced Ca2+ release in burst firing. An alternative mechanism could be that Ca2+ entry through L-type channels is sustained as the result of L-type channel phosphorylation by Ca2+-dependent protein kinases to support burst firing. The resulting increase in intracellular Ca2+ could activate Ca2+-sensitive protein kinases such as protein kinase C and calmodulin kinase II, both of which are capable of phosphorylating the L-type channels making them easier to open for longer period of time (Dzhura et al. 2000; Young & Yang, 2004; Yang et al. 2005).
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L-type channels have been found to mediate a slow excitatory synaptic drive to DA cells (Bonci et al. 1998). Their involvement in bursting could be via enhancing excitatory synaptic strength. This becomes a more important issue in the case of carbachol-induced bursting since there are known synaptic mechanisms involved in cholinergic excitation of DA cells (Mansvelder & McGehee, 2000; Mansvelder et al. 2002). However, synaptic blockade at the AMPA, NMDA and GABAA site did not alter carbachol-induced bursting, suggesting that in slices presynaptic elements do not carry active signals for cholinoceptors to modulate or different receptor subtypes to negate each others action at the terminal. Postsynaptically, activation of L-type channels may release DA from dendrites, which in turn terminates firing by D2-mediated autoinhibition. This autoinhibition may come in cycles similar to bursting because of somatodendritic DA reuptake mechanism. It has been shown that somatodendritic DA release is Ca2+ dependent and may contribute to DA cell bursting (Chen & Rice, 2001; Beckstead et al. 2004). It would be interesting to test whether this is the case for carbachol-induced bursting.
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How L-type channels operate at near-resting membrane potential to mediate burst firing may be explained by channel diversity and phosphorylation. There are reports that some L-type Ca2+ channels (Cav1.3, Cav1.4) are active at much more negative potentials than conventional high voltage-gated Ca2+ channels (Durante et al. 2004; Lipscombe et al. 2004; Power & Sah, 2005). In fact, small depolarizations preferentially open L-type channels on midbrain DA neurones (Durante et al. 2004), suggesting a more significant role for L-type channels in firing regulation. Alternatively, L-type channels may undergo Ca2+-dependent phosphorylation to facilitate opening (Dzhura et al. 2000).
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In conclusion, activation of cholinoceptors in the VTA leads to direct opening of L-type Ca2+ channels on the postsynaptic cell that gives rise to membrane oscillation and bursting. The involvement of L-type channels in DA cell bursting may represent a promising target for modulating the central DA system in disease conditions as supported by a finding that L-type blockers help relieve craving in drug addicts (Shulman et al. 1998).
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