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Departments of 1 Applied Therapeutics 2 Pharmacy Practice, Faculty of Pharmacy, Health Science Centre, Kuwait University, PO Box 24923, Safat 13110, Kuwait
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(Received 12 October 2003;
accepted after revision 9 December 2003;
first published online 12 December 2003)
Corresponding author S. B. Kombian: Department of Applied Therapeutics, Faculty of Pharmacy, Health Science Centre, Kuwait University, PO Box 24923, Safat 13110, Kuwait. Email: kombian{at}hsc.kuniv.edu.kw
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Introduction |
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The NAc is believed to serve as an interface where emotional events of limbic origin are converted into behavioural motor output. These behaviours are mediated by mesolimbic DA; a well recognized and extensively studied transmitter system with cell bodies located predominantly in the ventral tegmental area (VTA) (Koob & Bloom, 1988; Kuhar et al. 1991; Pennartz et al. 1994). CCK is abundant in this nucleus and has been reported to affect these DA-mediated behaviours (Crawley, 1988; Vaccarino, 1994). It is colocalized with DA arising from the VTA (Hokfelt et al. 1980) and substantia nigra (Lanca et al. 1998). A non-dopaminergic source from cortical areas has also been reported (Zaborszky et al. 1985; Seroogy & Fallon, 1989). CCK's innervations in NAc appear to follow a rostro-caudal pattern such that some subregions receive inputs from predominantly one source with minor contribution from other sources. In this regard, its inputs to the rostral NAc are from extra-mesencephalic regions (e.g. prefrontal cortex and amygdala; Gilles et al. 1983; Studler et al. 1985; Fallon & Seroogy, 1985; Zaborszky et al. 1985; Crawley, 1991) and mesencephalic structures (substantia nigra and the VTA; Lanca et al. 1998) while the caudal subregion receives its inputs primarily from the VTA (Zahm & Borg, 1992; Deutch, 1993; Zahm & Heimer, 1993; Lanca et al. 1998).
In addition to its presence in the NAc, both CCK receptors, CCKA and CCKB are also present (Carlberg et al. 1992) and there is evidence to suggest that these receptors also follow a rostro-caudal distribution (Crawley, 1992; Vaccarino, 1994; Mercer et al. 2000). CCKB receptors are predominantly localized on the somatodendrites and axons of the intrinsic GABAergic neurones (Berresford et al. 1987; Mercer et al. 2000). CCKA receptors on the other hand are present predominantly on dopaminergic (DAergic) afferent terminals in the NAC, since chemical lesioning of DAergic neurones results in marked reduction in the binding of a CCKA receptor ligand (Graham et al. 1991).
Pharmacological, neurochemical (White & Wang, 1984; Voigt et al. 1986; Marshall et al. 1991; Ferraro et al. 1996; Reum et al. 1997) and behavioural (De Witte et al. 1987; Dauge et al. 1989; Vaccarino & Rankin, 1989; Crawley, 1992) evidence indicate that CCK interacts with DA in the NAc to affect its function. However, most of the evidence shows that it either enhances or diminishes the DAergic system depending on whether it is applied to the rostral or caudal ends of the nucleus. Different receptors are reported to mediate these opposing effects (Voigt et al. 1986; De Witte et al. 1987; Marshall et al. 1991; Crawley, 1992; Reum et al. 1997).
In addition to the above CCK–DA interactions, CCK is also reported to increase or decrease the release of GABA in the NAc depending on the receptor subtype that is activated (Ferraro et al. 1996; Lanza & Makovec, 2000). In this nucleus, GABA is predominantly from axon collaterals arising from neighbouring projection cells (O'Donnell & Grace, 1993; Pennartz et al. 1994), as well as possible GABAergic interneurones (Meredith, 1999).
How this peptide interacts with all these transmitters to produce behavioural modifications is not clear. While the neurochemical and behavioural effects of CCK have been extensively studied, its cellular and synaptic effects are not well characterized. Electrophysiologically, DA is reported to act either directly on D1-like dopamine receptors located on glutamatergic terminals to decrease synaptic transmission (Harvey & Lacey, 1996; Nicola & Malenka, 1997) or indirectly on these same receptors located on medium spiny neurones to cause the generation of adenosine, which then acts on presynaptic glutamatergic terminals to decrease EPSCs (Harvey & Lacey, 1997; Buckby & Lacey, 2001; Kombian et al. 2003a). How CCK may influence these effects of DA is not known. The only evidence for any such interaction, to our knowledge, is from studies showing that iontophoretic application of CCK excited NAc cells in a dose-dependent manner (Wang et al. 1985; Wang, 1988). This excitation was similar to that produced by glutamate and could be reversed by DA. To better understand how CCK-induced neurochemical alterations in the NAc might translate into changes in cellular communication, we tested the effects of exogenous CCK on evoked excitatory postsynaptic currents recorded in NAc neurones. Furthermore, we examined the role that DA and GABA may play on any CCK effects.
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Methods |
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Slice preparation
Parasagittal forebrain slices containing the NAc and the cortex were generated using previously published techniques (Kombian & Malenka, 1994; see Fig. 1). Briefly, male Sprague-Dawley rats (75-150 g, 
3–5 weeks old) were anaesthetized with halothane and decapitated. The brain was quickly removed from the cranium and placed in ice-cold (4°C) artificial cerebrospinal fluid (ACSF) that was bubbled with 95% O2 and 5% CO2. The composition of the ACSF was (in mM): 126 NaCl; 2.5 KCl; 1.2 NaH2PO4; 1.2 MgCl2; 2.4 CaCl2; 18 NaHCO3; 11 glucose, producing a solution with osmolarity of between 310 and 320 mosmol l-1. The slices (350 µm thick) were cut in the ice-cold ACSF using OTS-4000 (Electron Microscopy Sciences, Pennsylvania, USA) or VT 1000S (Leica Microsystems, Wetzlar, Germany) tissue slicers. Slices were incubated in ACSF (bubbled continuously with 95% O2 and 5% CO2) at room temperature and allowed to recover for at least 1 h before experimentation.
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One brain slice was trimmed and transferred into a 500 µl capacity recording chamber and perfused submerged at a flow rate of 2–3 ml min-1 with ACSF that was bubbled continuously with 95% O2 and 5% CO2 at a temperature of 28–31°C. ‘Blind patch’ recordings were made from the rostral NAc (see Fig. 1) in the conventional whole-cell mode using glass electrodes with tip resistance of 4.0–8.0 M
. The internal recording solution had the following composition (in mM): 135 K-gluconate, 8 NaCl, 0.2 EGTA, 10 Hepes, 2 Mg-ATP, 0.2 GTP; pH and osmolarity adjusted to 7.3 (with KOH) and 270–280 mosmol l-1, respectively. Bipolar tungsten stimulating electrodes were positioned at the prefrontal cortex–accumbens border to evoke synaptic responses. Recordings were made using Axopatch 1D amplifiers (Axon Instruments Inc., Foster City, CA, USA) in either voltage or current clamp modes. Cells were voltage clamped at -80 mV (holding potential, Vh) and input (Rinput) and access (Ra) resistances of all cells were determined and monitored regularly throughout each experiment by applying a 20 mV hyperpolarizing pulse for 75 ms. Rinput was calculated from the steady-state current obtained during the pulse. The decay constant (
) of the capacitance transient was taken as a measure of Ra. All cells reported in this study had Ra values of 10–30 M. Data from cells that showed >15% changes in Ra during the experiment were excluded from further analysis.
All synaptic responses were recorded as inward currents at Vh of -80 mV. In control, the evoked synaptic response was a mixture of GABAA- and non-NMDA glutamate-receptor-mediated responses. Glutamate-induced, non-NMDA receptor-mediated pure EPSCs were isolated by applying 50 µM of picrotoxin, a GABAA receptor–chloride channel blocker. At this holding potential and in the presence of picrotoxin, the response was entirely non-NMDA receptor mediated, as it could be completely blocked by 6-7-dinitroquinoxaline-2,3-dione (DNQX; 10 µM). All cells had a graded evoked EPSC response to increasing stimulation intensity (ranging from 0.25 to 3.0 mA) and an intensity giving 50–60% of the maximum evoked synaptic response was used to evoke test responses. Steady-state current–voltage (I–V) curves were produced by an initial step change in the holding potential from -80 to 120 mV. The membrane potential was then slowly changed in a ramp fashion from -120 to -40 mV (in 18 s) before returning to the holding potential. The current produced in response to this ramped membrane potential was recorded to produce the I–V curve.
All data were acquired using pCLAMP Software (Clampex 7 or 8; Axon Instruments Inc.) at a sampling rate of 6.6 kHz and filtered at 1 kHz and stored for off-line analysis. Each stored trace was an average of two successive synaptic responses elicited at 10 s intervals. Hard copy chart records were also captured on Gould chart recorders (TA 240, Gould Instruments System Inc., Valley View, OH, USA) in some experiments.
Data analysis
EPSC amplitudes were measured from baseline to peak and taken as the excitatory synaptic strength at the chosen stimulus intensity. Responses were normalized by taking the mean of the last three or four responses prior to drug application and dividing the rest of the responses by this mean. These normalized values were then used for average plots. For these plots, all cells receiving the same treatment were aligned at the first minute of drug application and averaged over the entire period. All values are stated as mean ± standard error. One-way ANOVA and post hoc tests, as indicated in the Results section, were used to compare different values or treatments using SigmaStat® (Jandel Scientific Software, San Rafael, CA, USA). Significance was taken at the level of P 
0.05. Graphs were plotted using SigmaPlot® (Jandel Scientific Software, San Rafael, CA, USA), GraphPad Prism® (GraphPad Software Inc, San Diego, CA, USA) and CorelDraw® (Corel Corporation, Ottawa, ON, Canada).
Drug preparation and sources
All drugs were prepared and were bath perfused at final concentrations indicated by dissolving aliquots of stock in the ACSF. SCH23390and sulpiride were prepared daily and used within 24 h. Most routine laboratory chemicals as well as, 8-cyclopentyltheophylline (8-CPT), were from Sigma-Aldrich Chemie Gmbh (Steinheim, Germany). 6-7-Dinitroquinoxaline-2,3-dione (DNQX), SCH23390 sulpiride, proglumide were obtained from RBI (Natick, MA, USA); sulphated CCK octapeptide (CCK-8S), unsulphated CCK octapeptide (CCK-8US), LY225910, and CGP55845were from Tocris (Bristol, UK).
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Results |
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The results reported in this study were obtained from recordings in 78 cells located in the rostral pole of the core region of the NAc (Fig. 1). All cells were recorded very close to the stimulating electrode that was placed at the cortico-accumbens junction to activate prefrontal cortical excitatory afferents into this region (Fig. 1). By virtue of their relative abundance in the NAc, most of these recorded cells are likely to be medium spiny GABAergic neurones. These cells had passive and active membrane properties similar to those previously reported (Kombian & Malenka, 1994; Kombian et al. 2003a). All cells in this region had evoked responses composed of both glutamate- and GABA-mediated components. In the presence of picrotoxin 50 (µM) and at Vh of -80 mV, all of the evoked inward currents were glutamate-mediated excitatory postsynaptic currents (EPSCs) as they were completely blocked by DNQX (10 µM; data not shown but see Kombian et al. 2003a,b).
Effects of CCK on evoked EPSCs and NAc cells
Bath application of CCK-8S for 5–6 min in the presence of picrotoxin (50 µM) consistently caused a decrease in the amplitude of evoked EPSCs in 41 out of 44 (>90%) cells tested with CCK-8S alone. The onset of action was between 1 and 2 min with a peak effect in about 5–6 min (Fig. 2A). This CCK-8S-induced EPSC depression was concentration-dependent, with a maximum synaptic depression observed at 1 µM (-28.8 ± 1.6%, n= 8) and a calculated EC50 of 0.11 µM (Fig. 2B). This effect of CCK-8S tended to decrease in magnitude at concentrations higher than 1 µM. It was reversible, showing a recovery of 93.2 ± 7.3% after 10–15 min of washing out CCK-8S (Fig. 2A). Similar magnitudes of depression were obtained by repeated application of CCK-8S (1 µM) to the same cell following the washout period (-34.1 ± 11.1% in the first application compared to -32.9 ± 6.7% in the repeat application; n= 3 cells; P > 0.05, paired t test). In order to obtain a robust response that could be subjected to pharmacological characterization, 1 µM CCK-8S was employed for the rest of this study.
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In addition to the synaptic depression, both CCK-8S and CCK-8US induced an inward current in 90% of cells tested (19 out of 21 cells). CCK-8S induced an inward current that ranged from 5 to 30 pA (24.2 ± 9.2 pA; Fig. 3A) that recovered upon washout. CCK-8US, on the other hand, induced inward currents ranging from 10 to 50 pA (n= 8/10 cells), most of which did not recover as cells fired action potentials and were lost in the process. The inward current was accompanied by an increase in the input resistance of the cells (295.0 ± 24.6 M in control and 393.90 ± 38.6 M in CCK-8S; n= 8; and 474.0 ± 39.4 M in CCK-8US; n= 8; P < 0.05, one-way ANOVA; Fig. 3B). The steady-state I–V curve in the presence of CCK-8S intersected the curve produced in control at relatively negative potentials (-85 to -95 mV; Fig. 3C), producing an average estimated reversal potential (Erev) of -94.6 ± 3.0 mV (n= 4). In addition to these direct postsynaptic effects, CCK-8S also altered the kinetics of the evoked EPSC, causing a slowing in the decay rate resulting in an increase in the decay constant () of evoked EPSCs in 6 out of 8 cells (= 16.3 ± 1.8 ms versus 28.5 ± 1.0 ms in the presence of CCK-8S 1 µM; n= 6; P < 0.05, Fig. 3A). One of the other two cells showed a decrease in (while the other showed no change. In contrast to these postsynaptic actions of CCK-8S, we did not detect a change in the paired pulse ratio (PPR), a test often used to test for presynaptic actions of drugs (Manabe et al. 1993). The PPR was 1.2 ± 0.1 in control and 1.2 ± 0.1 in the presence of CCK-8S (n= 5, P > 0.05; paired t test; Fig. 4). These latter results suggest that CCK-8S does not act directly to cause a decrease in glutamate release.
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To verify that CCK-8S produced the above effects by activating CCK receptors present in the NAc (Innis & Snyder, 1980; Van Dijk et al. 1984; Gaudreau et al. 1985; Moran et al. 1986; Berresford et al. 1987; Mercer et al. 2000), we pretreated slices with proglumide (100 µM), a non-selective CCK receptor antagonist for 5–8 min and tested the effects of 1 µM CCK-8S. Cells, such as the one shown in Fig. 5A, that responded to CCK-8S with a robust synaptic depression no longer responded to CCK-8S in the presence of proglumide (-3.7 ± 1.7%, n= 5, P > 0.05 compared to control; Fig. 5). In addition, the postsynaptic current induced by CCK-8S was also blocked. Proglumide by itself did not produce significant changes in the postsynaptic holding current or synaptic response (-8.5 ± 13.2%, n= 5; P > 0.05 compared to control; paired t test; Fig. 5B and C). This effect of proglumide, combined with the fact that CCK-8US is known to selectively activate CCKB receptors (Innis & Snyder, 1980; Gaudreau et al. 1985), mimicking the postsynaptic and synaptic effects of the endogenous peptide CCK-8S (Figs 2 and 3), suggests that CCKB receptors mediate these effects of the endogenous peptide. To verify this, we used a potent selective CCKB receptor antagonist, LY225910 (Yu et al. 1991), in an attempt to block the CCK effect. Bath application of 100 nM LY225910 for 5–6 min caused no change in the holding current and evoked EPSC amplitude (-8.4 ± 4.3%, n= 6; P > 0.05, Fig. 6). When CCK-8S was subsequently applied in the presence of LY225910, it neither caused the predicted decrease in EPSC amplitude (5.5 ± 4.6%, n= 6, P > 0.05, unpaired t test, Fig. 6) nor induced the inward current. In three of these cells, following 10–15 min washout of LY225910 and CCK-8S, subsequent re-application of CCK-8S alone induced an inward current (Fig. 6A, insert) as well as causing a decrease in the evoked EPSC amplitude (-22.1 ± 6.5%; P < 0.05 compared to control; paired t test; Fig. 6A and C). These results indicate that CCK-8S produces both the postsynaptic inward current and excitatory synaptic depressant effects by activating CCKB receptors.
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Several lines of evidence indicate that CCK interacts with DA in the NAc to modulate the function of this nucleus (Voigt et al. 1986; De Witte et al. 1987; Dauge et al. 1989; Vaccarino & Rankin, 1989; Marshall et al. 1991; Crawley, 1992; Ferraro et al. 1996; Reum et al. 1997). In particular, neurochemical evidence indicates that CCK-8S increases DA outflow in the rostral NAc, the region in which the current studies were conducted, although another group observed the exact opposite (Marshall et al. 1991). Because, DA has been shown to depress EPSCs in this region via D1-like receptors (Pennartz et al. 1992; Nicola et al. 1996; Harvey & Lacey, 1996), we tested to see if CCK-8S produced the observed depression by utilizing DA as an intermediate. When cells were pretreated with 30 µM SCH23390 a DA D1-like receptor antagonist that has been shown to completely block DA's synaptic effects in this nucleus (Harvey & Lacey, 1996; Nicola et al. 1996), CCK-8S subsequently still produced a significant depression of -17.0 ± 6.5% (n= 5; P < 0.05 compared to control; paired t test; Fig. 7A and C). This level of depression was, however, less than that produced in the absence of SCH23390(-28.8 ± 1.6%; P < 0.05 compared to above depression, unpaired t test) obtained using the same batch of CCK-8S. This suggests that DA, acting on D1-like receptors, may contribute to the depressant effect of CCK-8S on evoked EPSCs. On the other hand, bath application of sulpiride (10 µM), a DA D2-like receptor antagonist predictably had no effect on the evoked EPSC. When CCK-8S (1 µM) was applied in the presence of sulpiride, it still produced a depression in evoked EPSC amplitude (-27.5 ± 9.6%, n= 4,P > 0.05 compared to the effect of the same batch of CCK-8S applied alone, unpaired t test; Fig. 7B and C).
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GABA mediates CCK-8S-induced depression of evoked EPSCs
Because CCK has been reported to increase the release of GABA in this subregion of the NAc (Lanza & Makovec, 2000), and GABA is known to depress EPSCs in this nucleus through GABAB receptors (Uchimura & North, 1991), it is possible that GABA, acting on GABAB receptors mediated the CCK-induced decrease in EPSC amplitude. To examine if GABA does indeed play a role in the CCK-8S-induced synaptic depression, we blocked GABAB receptors using CGP55845 a potent GABAB receptor antagonist (Davies et al. 1993; Lacey & Curtis, 1994). CGP55845(1 µM) by itself caused an enhancement in the evoked EPSC amplitude (56.7 ± 20.2%; n= 5; P < 0.05 compared to control, paired t test, Fig. 8), indicating a tonic action of GABA in depressing excitatory transmission in these cells. At the peak of the CGP55845effect, CCK-8S was applied and it no longer depressed the evoked EPSC amplitude (7.0 ± 8.1%; n= 5; P > 0.05 compared to control, paired t test, Fig. 8). However, in all five cells tested, CCK-8S still caused an inward current (26.2 ± 8.7 pA; n= 5; P > 0.05 compared to the CCK-8S-induced current in control, unpaired t test, Fig. 8A), suggesting that the synaptic depressant effect and the postsynaptic excitation (inward current) are produced by different mechanisms. Furthermore, in four additional cells, when the paired pulse protocol was applied in the presence of CGP55845(1 µM), this compound caused the expected increase in the evoked EPSC amplitude (58.7 ± 38.5%), which was accompanied by a predictable decrease in paired pulse facilitation (PPF) (1.8 ± 0.2 in control versus 1.2 ± 0.3 in CGP55845 P < 0.05; paired t test), suggesting that the GABAB receptors responsible for synaptic regulation in this nucleus are located on presynaptic glutamate terminals (see Fig. 9). Taken together, these results indicate that CCK-8S directly excites the medium spiny GABAergic neurones of the NAc to release GABA, which then acts on GABAB receptors located on glutamate terminals to decrease glutamate release and consequently depress the evoked EPSC.
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Discussion |
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CCKB receptors mediate CCK's cellular and synaptic effects
Both CCKA and CCKB receptors are present in the NAc (Carlberg et al. 1992; Mercer et al. 2000) and, as such, either one or both receptors may be activated by CCK to produce the above effects. The endogenously active CCK receptor ligand, CCK-8S caused a synaptic depression that was mimicked by CCK-8US, a ligand that binds preferentially only to CCKB receptors (Innis & Snyder, 1980; Gaudreau et al. 1985) and blocked by a selective CCKB receptor antagonist LY 225910 (Yu et al. 1991), indicating that CCK depresses excitatory synaptic transmission by activating CCKB receptors. The effect of CCK-8S on the EPSC amplitude peaked at a concentration of about 1 µM and tended to decline thereafter. This can arise from a possible activation of CCKA receptors that may have effects opposite to those of CCKB receptors or may be due to the desensitization of the CCKB receptors at higher concentrations (Burdakov & Galione, 2000).
In addition to these synaptic effects, CCK also induced an inward current (depolarization) in most cells in this region resulting in excitation and firing of action potentials. Furthermore, it changed the kinetics of the recorded non-NMDA receptor-mediated current, which could contribute to the decrease in amplitude of the recorded synaptic current. The mechanism by which CCK produces this effect on the non-NMDA receptor kinetics is yet to be determined but could involve changes in either the desensitization rate (Vyklicky et al. 1991) or channel conductance or both. These postsynaptic effects were direct, through the CCKB receptors, as they were blocked by CCKB, but not GABAB or DA receptor antagonists. Thus, while the postsynaptic (cellular) effects of CCK-8S were produced directly through the activation of CCKB receptors, the synaptic depressant effect is more complicated, involving (a) a possible direct postsynaptic action of CCK on CCKB receptors and (b) an indirect action through GABA, and possibly DA.
Postsynaptic actions of CCK in NAc cells
The inward current induced by both CCK-8S and CCK-8US in NAc neurones resulted in their excitation as they fired action potentials that were superimposed on the evoked EPSC. This is in agreement with previous in vivo reports that showed that CCK excited NAc cells leading to increased single unit activity (Wang et al. 1985). This inward current was accompanied by an increase in the input resistance (Rinput) recorded around the resting potentials of these cells. This increase in Rinput suggests that a resting current was closed to produce the inward current. As these cells rest at very negative potentials (around -80 mV), the only currents that are active at such potentials are those carried by potassium. Furthermore, the very negative estimated reversal potential of the CCK-8S-induced current suggests that it is a potassium current. Thus, CCK-8S closes one or more potassium currents to depolarize these cells. This action of CCK-8S is via CCKB receptors which have been shown immunohistochemically to be present on somatodendrites of NAc cells (Mercer et al. 2000). This action of CCK contrasts with its action in the arcuate nucleus where it does not induce any current but instead potentiates A-currents to slow down the firing of these cells (Burdakov & Ashcroft, 2002). Interestingly, this effect was also produced through the activation of postsynaptic CCKB receptors, the same receptors that we observed here to cause the closure of this as-yet-uncharacterized potassium channel(s). It is important to know the nature of this potassium current as we strive to understand the actions of this peptide in the NAc and the CNS in general. In addition to the above, CCK-8S also slowed down the kinetics of the non-NMDA glutamate receptor-mediated EPSC. The increase in the decay constant () suggests that CCK directly interacts with this channel to either decrease desensitization (Vyklicky et al. 1991) or affect other channel kinetics and this may be responsible, at least in part, for the decrease in the EPSC amplitude.
GABA, and to a lesser extent DA, mediates CCK-induced synaptic depression
The reported co-localization and interaction of CCK with DA in the NAc to influence several behaviours (Vaccarino & Koob, 1984; De Witte et al. 1987; Vaccarino & Rankin, 1989; Crawley, 1992) suggests that, at least, some of CCK's effects on cellular and synaptic responses and conductances in this nucleus may be mediated through DA. Indeed, while we found that CCK's effect on EPSC was slightly attenuated by SCH23390 a DA D1-like receptor antagonist that has been shown to block DA synaptic effects in this nucleus (Pennartz et al. 1992; Harvey & Lacey, 1996; Nicola et al. 1996), this blocking effect of SCH23390was incomplete as CCK still caused a statistically significant depression of the evoked EPSC, albeit less than in control. This may mean that CCK does not rely strongly on DA to mediate its synaptic effects, or it may also reflect the well-documented opposing actions of CCK on DA release (Voigt et al. 1986; Lane et al. 1986; Ruggeri et al. 1987; Vickroy & Bianchi, 1989) whereby the opposing actions of CCKA and CCKB receptor activation can lead to a minimal change in DA release (Hamilton et al. 1984; Marshall et al. 1991). While it is possible for CCK to modulate the release of DA in vivo to affect DA-dependent behaviours, the finding here suggests that the main action of CCK in modulating these behaviours in the NAc (Vaccarino & Koob, 1984; De Witte et al. 1987; Vaccarino & Rankin, 1989; Crawley, 1992) may be direct, by influencing the response of NAc cells to DA rather than through the release or blockade of DA release. Even if the release of DA plays an important role in CCK actions, the level of activation of CCKA and CCKB receptors would have to be fine tuned to offset the balance in favour of one or the other. How this fine-tuning may be attained in vivo remains to be determined but the differential distribution of CCK-containing terminals and receptors in the different subregions of the NAc may allow for selective regional release and activation of only one type of receptor.
Further to this DA–CCK interaction, CCK has also been reported to increase the release of GABA in the rostral NAc (Lanza & Makovec, 2000). The depolarization and the resultant action potential firing observed above would cause an increase in the release of GABA from terminals of axon collaterals. The released GABA can act on appropriate receptors, usually GABAB receptors, to cause depression of excitatory synaptic transmission (Uchimura & North, 1991). These GABAB receptors are located on presynaptic glutamate terminals and their activation leads to a decrease in glutamate release and hence a decrease in the EPSC amplitude. This presynaptic locus of action of GABA to depress the EPSC is inferred from the observation that CGP55845caused an increase in the evoked EPSC amplitude, an effect that was accompanied by a change (decrease) in paired pulse facilitation; a mainly presynaptic phenomenon (Manabe et al. 1993; Zucker, 1989). Our finding that CGP55845 the GABAB receptor antagonist, completely blocked the CCK-8S-induced synaptic effects, but not the inward current, indicates that CCK mainly employs GABA to mediate synaptic depression in this nucleus. This is in agreement with a recent report by Lanza & Makovec (2000) that CCK, in contrast to its opposing effect on DA release, causes only an increase in the release of GABA in the rostral NAc (but see Ferraro et al. 1996).
An intriguing finding in this study was that, despite the reported possible contribution of DA to the CCK-induced synaptic depression, blockade of GABAB receptors produced a complete block of the CCK effect, suggesting that this is the main mechanism responsible for the CCK-induced synaptic depression. This may happen if GABA's effect possibly overwhelms the contributions of direct postsynaptic and DAergic mechanisms to the CCK-induced synaptic depression. Alternatively, it may also indicate that DA produces some of its effect in the NAc indirectly through the release of GABA. This possibility needs to be further examined as it may reveal yet another novel mechanism by which DA produces synaptic depression in this nucleus (see Harvey & Lacey, 1997). Another intriguing finding was that, although both GABA and DA are widely known, and are reported in this nucleus to depress synaptic transmission by presynaptic mechanisms (Uchimura & North, 1991; Harvey & Lacey, 1996), we did not see a change in PPR, a paradigm often used to test for the presence of presynaptic action of substances in synaptic physiology (Zucker, 1989; Manabe et al. 1993; Kombian et al. 2003a). Our inability to detect a change in PPR in this case may be due to the reported inability of PPR to detect presynaptic effects when changes in synaptic responses are not greater than 60% of the initial response (Manabe et al. 1993). It may also be a consequence of the combined pre- and postsynaptic actions of CCK that mask any possible changes in PPR.
This study thus reveals that the effect of CCK in the NAc is to excite the projection medium spiny GABAergic neurones directly through the activation of CCKB receptors which are present on these cells. This excitation results in the release of GABA which is the main mediator of CCK-induced synaptic depression (Fig. 9). The minimal effect of DA observed here may reflect the well-documented opposing actions of CCK on DA release in this nucleus (see Introduction). The physiological significance of directly exciting NAc cells while depressing glutamate-mediated excitation is not yet clear to us. The obvious benefit of such a dual action would be to prevent or reduce excessive excitation of NAc neurones, especially if the direct postsynaptic excitation precedes excitatory synaptic depression (see Fig. 9B). This may be the case, as the inward current was usually observed first and peaked before the peak of the synaptic depression. The CCK-induced inhibition of excitatory synaptic transmission may help to ensure that afferent (cortical) control of NAc output is minimized while local intra-accumbal control is optimized (Fig. 9A). Since CCK itself does not appear to have a direct presynaptic effect on glutamate release (see Lanza & Makovec, 2000), its ability to select and dampen certain excitatory inputs would be limited except through intermediate modulators such as GABA.
Functionally, the NAc is thought to filter out competing afferent excitatory signals allowing only appropriate ones through. This enables animals to focus on only certain tasks at any particular time. If released CCK excites NAc projection neurones indiscriminately, then this ability of the NAc would be lost. This would lead to an inability to concentrate or focus on appropriate or relevant tasks and behaviours while ignoring irrelevant ones, a characteristic seen in schizophrenics. In this regard, it has been reported that latent inhibition in rats, an animal model that is relevant to schizophrenia, is modulated by CCK receptor antagonists (Feifel & Swerdlow, 1997; Gracey et al. 2000, 2002) suggesting that CCK over-activity may be involved in the pathophysiology of schizophrenia and other psychiatric disorders (Tachikawa et al. 2001; Hattori et al. 2001; Wang et al. 2002; De Wied & Sigling, 2002). Indeed, CCK receptor antagonists are being developed and evaluated as antipsychotic agents (Feifel & Swerdlow, 1997; Feifel et al. 1999). The selective action of such drugs in the NAc in altering synaptic and cellular excitability induced by CCK may serve as the basis for their therapeutic action.
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References |
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