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Neuroscience |
1 Department of Pharmacology, Physiology and Therapeutics, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA
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(Received 25 December 2005;
accepted after revision 2 February 2006;
first published online 2 February 2006)
Corresponding author S. Lei: Department of Pharmacology, Physiology and Therapeutics, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA. Email: slei{at}medicine.nodak.edu
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Introduction |
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The effects of CCK are mediated by two types of G protein-coupled receptors: CCK-A and CCK-B receptors (Jagerschmidt et al. 1995; Noble & Roques, 1999; Pommier et al. 1999, 2003). Whereas CCK-A receptors are present in peripheral tissues and a few discrete brain regions (Moran et al. 1986; Hill et al. 1987, 1990), CCK-B receptors are the predominant form found in the brain (Van Dijk et al. 1984). Both receptors are coupled to phospholipase C (PLC), leading to increases in intracellular Ca2+ release and activation of protein kinase C (PKC; Wank, 1995). In addition, CCK-A receptor activation increases adenylyl cyclase activity, which further enhances the generation of cyclic AMP and subsequent activation of protein kinase A (Wank, 1995), whereas CCK-B receptors elevate phospholipase A2 (PLA2) activity, resulting in the release of arachidonic acid (Pommier et al. 1999, 2003). These intracellular signals may be involved in the effects of CCK.
Cholecystokinin is extensively expressed in the hippocampal formation. Cholecystokinin immunoreactive fibres are located around the cell bodies in the entorhinal cortex, subiculum and stratum pyramidale of Ammon's horn, and among the granule cells and inner molecular layer of the dentate gyrus (Greenwood et al. 1981). The CCK immunoreactive neurons appear to be non-pyramidal cells and may represent a subpopulation of GABAergic interneurons (Somogyi et al. 1984; Hendry & Jones, 1985; Nunzi et al. 1985). In addition to containing CCK, the hippocampal formation also expresses CCK receptors. The dentate gyrus exhibits the highest levels of binding sites for CCK, and moderate to light labelling has been observed in the stratum pyramidale of CA3 and CA1 areas, respectively (Kritzer et al. 1988; Kohler & Chan-Palay, 1988). The type of CCK receptors in the hippocampus is CCK-B (Shigeyoshi et al. 1994).
The selective distribution of CCK in GABAergic interneurons and the wide expression of CCK-B receptors in the hippocampal formation suggest that CCK modulates GABAergic functions in the hippocampal formation. Indeed, CCK has been reported to increase GABA release from hippocampal slices (Perez de la Mora et al. 1993) and enhance GABAA receptor-mediated IPSCs in the CA1 region (Miller et al. 1997), although two studies suggest that CCK does not change GABA release from rat hippocampal synaptosomes (Breukel et al. 1997) and hippocampal slices (Migaud et al. 1994). To solve these controversies and to determine the involved cellular and molecular mechanisms, we initially examined the effects of CCK on GABAergic synaptic transmission in the hippocampal formation. We then explored the underlying mechanisms by focusing on the dentate gyrus of the hippocampus because the highest density of CCK receptors is detected in this region (Kritzer et al. 1988; Kohler & Chan-Palay, 1988). Our results demonstrate that CCK has a bidirectional regulation of GABAergic transmission: an initial transient increase followed by a persistent reduction in GABA release. The CCK-mediated transient increase in GABA release is related to the inhibition of Ca2+-activated K+ channel activity. The effects of CCK on GABA release are not related to PLC or PLA2 pathway, suggesting a direct coupling of G-proteins to Ca2+-activated K+ channels. Our results provide a novel cellular mechanism to explain the functions of CCK in the brain.
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Methods |
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Horizontal hippocampal slices (400 µm) including the entorhinal cortex, subiculum and hippocampus were cut using a Vibratome (Leica VT1000S) usually from 10- to 20-day-old Sprague–Dawley rats as previously described (Lei & McBain, 2003). For those experiments examining the development of CCK-mediated modulation of GABA release, we expanded the age of the rats to 6- and 31-day-old-rats. Rats were deeply anaesthetized with isoflurane, rapidly decapitated, and the brain was dissected out in ice-cold saline solution that contained (mM): 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 5.0 MgCl2 and 10 glucose, saturated with 95% O2–5% CO2, pH 7.4. Slices were initially incubated in the above solution at 35°C for 40 min for recovery and then kept at room temperature until use. All animal procedures conformed to the guidelines approved by the University of North Dakota Animal Care and Use Committee.
Recordings of spontaneous, miniature and evoked GABAA receptor-mediated IPSCs
Whole-cell patch-clamp recordings using an Axopatch 200B or a Multiclamp 700B (Axon Instruments, Union City, CA, USA) in voltage-clamp mode were made usually from dentate gyrus granule cells visually identified with infrared video microscopy (Olympus BX51WI) and differential interference contrast optics unless stated otherwise. For the experiments examining the effects of CCK on GABAergic transmission in different regions of the hippocampal formation, we recorded from neurons in the entorhinal cortex and subiculum. The recording electrodes were filled with the following (mM): 100 caesium gluconate, 0.6 EGTA, 5 MgCl2, 8 NaCl, 2 ATP2Na, 0.3 GTPNa, 40 Hepes and 1 QX-314, pH 7.2–7.3 (adjusted with CsOH). The extracellular solution comprised the following (mM): 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1.5 MgCl2, 2.5 CaCl2 and 10 glucose, saturated with 95% O2–5% CO2, pH 7.4. To record GABAA receptor-mediated spontaneous IPSCs (sIPSCs), the external solution was supplemented with DL-2-Amino-5-phosphono-pentanoic acid (DL-APV; 100 µM) to block NMDA receptor-mediated responses and 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 10 µM) to block AMPA receptor-mediated responses. Under these conditions, the recorded inhibitory currents had a reversal potential of approximately –30 mV and were completely blocked by bicuculline methobromide (10 µM), confirming that they were mediated by GABAA receptors. Usually sIPSCs were recorded at a holding potential of +30 mV (Lei & McBain, 2003). Miniature IPSCs (mIPSCs) were recorded by including TTX (0.5 µM) in the above external solution to block action potential-dependent responses. Evoked IPSCs were recorded from dentate gyrus granule cells using the same internal and external solution by placing a stimulation electrode in the hilus. Data were filtered at 2 kHz, digitized at 10 kHz and acquired on-line using pClamp 9 (Clampex) software (Axon Instruments). The recorded sIPSCs and mIPSCs were subsequently analysed by Mini Analysis 6.0.1 (Synaptosoft Inc., Decatur, GA, USA). Each detected event was inspected visually to exclude obvious artifacts before analysis. Mean amplitude, frequency, cumulative amplitude and frequency histograms were calculated by this program. The recorded evoked IPSCs were analysed by pClamp 9 (Clampfit). Cholecystokinin was applied via the bath. To avoid desensitization induced by repeated applications of CCK, one slice was limited to only one application of CCK.
Recordings of action potentials
Spontaneous action potential firing was recorded from interneurons in the hilus with whole cell patch-clamp recordings in current-clamp mode. Caesium gluconate in the above intracellular solution was replaced with the same concentration of potassium gluconate, and QX-314 was omitted. Because dialysis of K+-containing internal solution into the cells can change the resting membrane potential and influence the spontaneous action potential firing, we waited for 
15 min after the formation of whole-cell recordings to allow the resting membrane potential to stabilize. Data were obtained only from those cells displaying resting membrane potentials negative to –60 mV. Usually, for most of the cells a positive current injection was needed to elevate the resting membrane potential to approximately –45 mV to induce spontaneous action potential firing. Cholecystokinin was applied after the action potential firing had been stable for 5–10 min. The frequency of the action potentials was calculated by Mini Analysis 6.0.1.
Construction of voltage–current curves and recordings of Ca2+ channel currents
Voltage–current curves were constructed from the interneurons in the hilus. Potassium gluconate internal solution was used, and the external solution contained TTX (0.5 µM) to block Na+ channels. Voltage–current relationships were obtained by using a ramp protocol from –100 to +40 mV at a speed of 0.08 mV ms–1. Calcium channel currents were recorded from the interneurons in the hilus. The external solution contained TTX (0.5 µM) to block Na+ channels. The pipette solution contained (mM): 100 Cs-gluconate, 30 tetraethylammonium, 1 CaCl2, 1 MgCl2, 4 ATP, 0.3 GTP, 5 EGTA and 10 Hepes (pH adjusted to 7.2 with CsOH). Leak currents were subtracted using P/N leak subtraction in Clampex.
Outside-out nucleated patch recordings
Outside-out nucleated patch recordings from interneurons in the hilus were carried out as described by Lei et al. (2001). The pipettes contained potassium gluconate internal solution, and the external solution contained TTX (0.5 µM). After formation of a whole-cell recording, a piece of membrane was excised by slowly pulling the electrode away from the patched interneuron with a slight negative pressure inside the pipette. Voltage–current relationship was then obtained from the excised patches before and during the application of CCK by using the ramp protocol from –100 to +40 mV at a speed of 0.08 mV ms–1.
Data analysis
Data are presented as the means ±S.E.M. Student's paired or unpaired t test or analysis of variance (ANOVA) was used for statistical analysis as appropriate; P values are reported throughout the text and significance was set as P < 0.05.
Chemicals
CCK-8S, CCK-8U and CCK-4 were purchased from American Peptide Company. YM 022 was the product from Tocris. Lorglumide, SKF96365, U73122, thapsigargin, calphostin C and PACOCF3 were purchased from BIOMOL. The other chemicals were from Sigma.
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Results |
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Although CCK has been reported to increase GABA release from rat cortical slices (Ferraro et al. 1999), rat nucleus accumbens (Lanza & Makovec, 2000) and the cerebral cortex of freely moving rats (Siniscalchi et al. 2003), there are inconsistent results as to whether CCK modulates GABA release in the hippocampus (Perez de la Mora et al. 1993; Migaud et al. 1994; Breukel et al. 1997; Miller et al. 1997). Since most of the studies were conducted by measuring GABA concentration in the perfusate of either hippocampal slices or hippocampal synaptosomes, which may represent a neutralized effect of CCK in all regions of the hippocampal formation, we studied the effects of CCK on GABAergic synaptic transmission by recording GABAA receptor-mediated spontaneous IPSCs from different regions of the hippocampal formation to solve these controversies. We initially examined the effects of CCK on sIPSCs recorded from dentate gyrus granule cells because the highest density of CCK receptors has been detected in the dentate gyrus (Kritzer et al. 1988; Kohler & Chan-Palay, 1988). Bath application of the sulphated octapeptide (CCK-8S, 0.1–1 µM) produced a bidirectional modulation of sIPSC frequency recorded from dentate gyrus granule cells. Cholecystokinin transiently increased, and then gradually reduced sIPSC frequency (Fig. 1A, B, C and E). Spontaneous IPSC frequency usually increased to a maximum in 3–4 min after the beginning of CCK application (increased by 139 ± 37% at 0.5 µM, n= 11, P= 0.004; Fig. 1A, B and E) and then began to decline (Fig. 1B). Cholecystokinin-induced gradual decline of sIPSC frequency is probably due to the agonist-induced desensitization of CCK receptors, because these receptors undergo considerable desensitization (Shinohara & Kawasaki, 1994). At the end of CCK application (0.5 µM, 20 min), sIPSC frequency was reduced to 55 ± 8% of the initial value before CCK application (n= 11, P= 0.0002). The depressant effect of CCK was irreversible and became even more conspicuous after washing in CCK-free external solution. For 10 min after washing, sIPSC frequency was further reduced to 35 ± 9% of control values (n= 11, P= 0.00003, Fig. 1B and E). For five of these cells, sIPSC frequency did not recover even after 30 min washing (15 ± 6% of control, n= 5, P= 0.0001, data not shown), suggesting that CCK-induced persistent depression is irreversible. Cholecystokinin-mediated change in sIPSC amplitude was not consistent among cells, varying from an increase (Fig. 1D) through no change to slight reduction. It appeared that the effects of CCK on sIPSC amplitude were dependent on the initial frequency of sIPSCs. For the cells showing low initial sIPSC frequency, CCK usually increased sIPSC amplitude. However, for those cells displaying high initial sIPSC frequency, there was a temporal summation of sIPSCs (the increased sIPSCs overlapped each other) and the actual sIPSC amplitude was either unchanged or slightly reduced. The summarized data indicate that there is no significant difference in CCK-mediated change in sIPSC amplitude (Fig. 1F).
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Although CCK-8S is the major form of CCK in the brain (Rehfeld et al. 1985; You et al. 1994), we also examined the effects of the unsulphated CCK octapeptide (CCK-8U) and the tetrapeptide (CCK-4). Application of CCK-8U (0.5 µM) transiently increased sIPSC frequency to 228 ± 44% of control values (n= 7, P= 0.03) followed by a persistent reduction (30 ± 9% of control values, n= 7, P= 0.0002, Fig. 3A). Application of CCK-4 (0.5 µM) also increased sIPSC frequency to 289 ± 31% of control values (n= 9, P= 0.0003) within 4 min of its application followed by a reduction (40 ± 7% of control values, n= 9, P < 0.0001, Fig. 3B). Because CCK-8U is a weak agonist for CCK-A receptors, but it is almost as potent as CCK-8S for CCK-B receptors (Wank, 1995), these results suggest that the effects of CCK on modulating sIPSC frequency are mediated by the activation of CCK-B receptors. To further identify the involved type of CCK receptors, we used the selective antagonists for CCK-A or CCK-B receptors. Application of the selective CCK-B receptor inhibitor, YM 022 (1 µM, Nishida et al. 1994), alone did not significantly alter sIPSC frequency (100 ± 6% of control values, n= 7, P= 0.98, Fig. 3C). In the continuous presence of YM 022, application of CCK failed to significantly increase sIPSC frequency (89 ± 12% of control values, n= 7, P= 0.38, Fig. 3C), suggesting that CCK-B receptors are required for CCK-mediated modulation of sIPSCs. We also used a selective CCK-A receptor inhibitor, lorglumide (1 µM, de Tullio et al. 1999) to examine the potential roles of CCK-A receptors. However, in the presence of lorglumide (1 µM), CCK still transiently increased sIPSC frequency to 320 ± 61% of control values (n= 5, P= 0.02, Fig. 3D) followed by a reduction (40 ± 8% of control values, n= 5, P= 0.002, Fig. 3D), suggesting that CCK-A receptors are not involved. All these results unanimously demonstrate that the effects of CCK on sIPSCs are mediated by the activation of CCK-B receptors. This conclusion is also consistent with the previous observation that the CCK receptors in the hippocampus are CCK-B receptor subtype (Shigeyoshi et al. 1994). Because the sulphated CCK is the major form of CCK in the brain (Rehfeld et al. 1985; You et al. 1994), we used the sulphated CCK (CCK-8S) for the rest of the experiments.
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Several hypotheses could be proposed to explain CCK-mediated initial increases in sIPSCs, as follows: (1) CCK facilitates the generation of action potentials to increase the excitability of GABAergic interneurons to increase GABA release; (2) CCK increases Ca2+ channel activity to increase GABA release; (3) CCK modulates the exocytosis machinery downstream of Ca2+ influx; and (4) CCK increases the functions of postsynaptic GABAA receptors. We recorded the miniature IPSCs (mIPSCs) in the presence of TTX and the evoked IPSCs to test these hypotheses. Cholecystokinin (0.5 µM) had no effects on either the frequency (control, 0.59 ± 0.18 Hz; CCK, 0.57 ± 0.15 Hz, n= 6, P= 0.69, Fig. 4A, B and C) or the amplitude (control, 21.8 ± 1.3 pA; CCK, 20.3 ± 1.3 pA, n= 6, P= 0.09, Fig. 3A, B and D) of mIPSCs recorded in the presence of TTX (0.5 µM). Since mIPSCs recorded in the presence of TTX are generally considered to be action potential- and Ca2+-independent, this result suggests that CCK has no effects on either postsynaptic GABAA receptors or exocytosis downstream of Ca2+ influx. Moreover, if CCK acts by directly increasing Ca2+ channel activity, it should equally increase the evoked IPSCs because voltage-gated Ca2+ channels are functional for the evoked IPSCs. We recorded from dentate granule cells GABAA receptor-mediated IPSCs evoked by placing a stimulation electrode in the hilus. However, application of CCK (0.5 µM) did not significantly change the amplitude of the evoked IPSCs (103 ± 15% of control values, n= 5, P= 0.87, Fig. 5), suggesting that CCK has no effects on presynaptic Ca2+ channels. Because action potentials underlying the evoked IPSCs are generated by exogenous stimulation-induced depolarization, whereas those responsible for spontaneous IPSCs are determined by the intrinsic excitability of neurons, the results that CCK modulates sIPSC frequency without altering the evoked IPSC amplitude suggest that CCK alters the excitability of GABAergic interneurons and regulates the generation of action potentials.
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We then tested the hypothesis that CCK modulates GABA release via regulating the excitability (generation of action potentials) of GABAergic interneurons. We directly recorded spontaneous action potentials from the interneurons in the hilus. Caesium gluconate in the recording pipettes was replaced by the same concentration of potassium gluconate, and QX-314 was omitted. For most of the interneurons, application of CCK resulted in a transient increase, followed by a reduction in the frequency of action potential firing (Fig. 6A). However, there were still some interneurons in which the CCK-mediated late phase of reduction in action potential firing was not evident. The summarized data are shown in Fig. 6B and D. Cholecystokinin increased the spontaneous action potential firing frequency to 523 ± 164% of control values (n= 13, P = 0.02, Fig. 6A, B and D). While the action potential firing frequency at the end of CCK application was not significantly reduced to lower than the initial value (CCK after 20 min, 90 ± 39% of control, n= 13, P= 0.8, Fig. 6B), the action potential firing frequency was significantly lower than control values after 10 min washing in CCK-free external solution (53 ± 17% of control values, n= 13, P= 0.02, Fig. 6B and D). The reason underlying the CCK-mediated obvious late phase of depression for sIPSC frequency (Fig. 1B) versus the slight late phase of depression for action potential firing (Fig. 6B) may be that the former is the synchronized results from many interneurons whereas the later is the action of a single interneuron. Nonetheless, these results suggest that CCK regulates GABA release by modulating action potential firing of the interneurons. To detect the changes in action potential shape, we averaged the action potentials recorded before and during the application of CCK (Fig. 6C). Cholecystokinin significantly reduced the amplitudes of both the spike (92 ± 2% of control values, n= 9, P= 0.004, Fig. 6C and D) and the after-hyperpolarization (AHP; 71 ± 7% of control values, n= 9, P= 0.0004, Fig. 6C and D). The CCK-mediated slight reduction in spike amplitude may be due to the inactivation of Na+ channels, because a high frequency of firing is likely to depolarize the membranes and inactivate Na+ channels. The result that CCK reduces the amplitude of the AHP suggests that CCK increases action potential firing frequency by facilitating the achievement of the threshold to fire another action potential.
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The amplitude of the AHP of the action potentials is determined by Ca2+-activated K+ channels (Storm, 1987). The result that CCK attenuated the amplitude of the AHP suggests that CCK depresses IK(Ca). We therefore next examined the effects of CCK on K+ channels of the interneurons in the hilus. We constructed the voltage–current relationship of the interneurons in the presence of TTX (0.5 µM) by applying a ramp protocol (Fig. 7A). Application of CCK (0.5 µM) had no conspicuous effects on the V–I curves at voltages close to or more negative than the resting membrane potential, but significantly reduced the outward current at depolarizing voltages (Fig. 7A). The amplitude of the current at +30 mV was significantly reduced from 1.09 ± 0.15 nA in control conditions to 0.72 ± 0.09 nA (n= 8, P= 0.006) by 3–5 min after the beginning of CCK application. After washing for 10 min in CCK-free external solution, the outward current was slightly but significantly higher than the initial control conditions (control, 1.09 ± 0.15 nA; wash, 1.18 ± 0.16 nA, n= 8, P= 0.02, Fig. 7A). This phenomenon (over-recovery) has also been observed for M-channels after inhibition by muscarine (Marrion et al. 1991; Chen et al. 1993; Tokimasa et al. 1996; Kurennyi et al. 1997), suggesting that some K+ channels share the same properties. We considered that the over-recovery of the outward K+ channels after CCK-mediated inhibition was at least part of the mechanism underlying the CCK-induced late phase of reduction (see Discussion). Cholecystokinin-mediated modulation of the outward current was due to the inhibition of IK(Ca) because when the extracellular solution was switched to a solution containing 0 Ca2+ but 100 µM Cd2+ to block Ca2+ channels, CCK no longer inhibited the outward currents, although the outward current was reduced in this condition (0 Ca2++ 100 µM Cd2+, 66 ± 2% of control values; 0 Ca2++ 100 µM Cd2++ CCK, 67 ± 3% of control values, n= 6, P= 0.6, Fig. 7B). To further confirm the involvement of IK(Ca), we used IK(Ca) inhibitors. Application of the small-conductance IK(Ca) inhibitor, apamin (100 nM), did not apparently change the V–I curve (at +30 mV: control, 1.41 ± 0.13 nA; apamin, 1.40 ± 0.12 nA, n= 7, P= 0.53, Fig. 7C), nor did it block CCK-mediated inhibition of the outward currents (apamin, 1.40 ± 0.12 nA; apamin + CCK, 1.02 ± 0.13 nA, n= 7, P= 0.004, Fig. 7C), suggesting that the small-conductance IK(Ca) are not involved in the effects of CCK on the outward currents. We then used the large-conductance IK(Ca) inhibitors: paxilline and charybdotoxin. Application of paxilline (10 µM) alone significantly reduced the outward current (control, 898 ± 71 pA; paxilline, 635 ± 89 pA, n= 7, P= 0.0003, Fig. 7D), followed by a slight reduction in the presence of CCK (paxilline, 635 ± 89 pA; paxilline + CCK, 587 ± 85 pA, n= 7, P= 0.04, Fig. 7D). Application of the large-conductance IK(Ca) inhibitor charybdotoxin (50 nM) alone significantly reduced the outward current (control, 876 ± 40 pA; charybdotoxin, 597 ± 36 pA, n= 5, P= 0.0001, data not shown). Subsequent application of CCK in the presence of charybdotoxin failed to significantly inhibit the outward currents further (charybdotoxin, 597 ± 36 pA; charybdotoxin + CCK, 588 ± 29 pA, n= 5, P= 0.38, data not shown). The slightly different effects of CCK in the presence of paxilline or charybdotoxin may be because of their distinct specificities for IK(Ca). Nevertheless, these results suggest that CCK modulates GABA release by regulating the large-conductance IK(Ca) in interneurons.
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Cholecystokinin has also been reported to inhibit K+ channels responsible for the control of resting membrane potentials (Branchereau et al. 1993; Cox et al. 1995; Miller et al. 1997) and to open cation channels to generate membrane depolarization (Wu & Wang, 1996a, b; Wang & Sims, 1998; Chakfe & Bourque, 2000, 2001). Either of these mechanisms would depolarize the membrane to change the holding current when the cells are held at voltages close to the resting membrane potentials. We next tested whether these mechanisms are involved by recording the holding currents at –55 mV, a potential close to the resting membrane potential. The extracellular solution contained (µM): 0.5 TTX, 10 DNQX, 100 DL-APV and 10 bicuculline. However, CCK failed to change the holding currents (n= 8, P= 0.28, Fig. 7G), suggesting that it is unlikely that CCK-mediated increases in GABA release are caused by a change in the resting membrane potentials. We also tested the roles of receptor-operated cation channels by applying SKF 96365, a broadly used blocker of cation channels (Merritt et al. 1990). This compound blocks CCK-A receptor-mediated inward current (Tsujino et al. 2005). Application of SKF 96365 (50 µM) alone did not significantly change sIPSC frequency (94 ± 6% of control values, n= 6, P= 0.3, Fig. 7H), nor did it block the effects of CCK on sIPSC frequency (CCK at 3 min, 365 ± 9% of control values, n= 6, P= 0.03; CCK at 20 min, 61 ± 13% of control values, n= 6, P= 0.03, Fig. 7H), suggesting that cation channels are unlikely to be involved. These results suggest that CCK-mediated increases in GABA release are unlikely to be caused by an inhibition of K+ channels responsible for the resting membrane potentials or opening of cation channels in the dentate gyrus region.
Inhibition of IK(Ca) inhibits the effects of CCK on GABA release
If IK(Ca) channels are responsible for CCK-mediated modulation of GABA release, application of IK(Ca) inhibitors should block or at least reduce the effects of CCK on sIPSC frequency. Therefore, we next tested the effects of IK(Ca) inhibitors on CCK-mediated changes in sIPSC frequency. Application of apamin (100 nM) alone did not significantly alter sIPSC frequency (90 ± 8% of control values, n= 7, P= 0.27, Fig. 8A) nor did it block the effects of CCK on sIPSCs (CCK at 3 min, 323 ± 69% of control values, n= 7, P= 0.02; wash at 10 min, 60 ± 9% of control values, n= 7, P= 0.004, Fig. 8A), suggesting that the small-conductance IK(Ca) is not involved in the effects of CCK on GABA release. However, application of paxilline (10 µM) significantly increased sIPSC frequency to 142 ± 9% of control values (n= 10, P= 0.001, Fig. 8B), suggesting that the large-conductance IK(Ca) channels are involved in modulating GABA release. Furthermore, in the presence of paxilline, application of CCK only slightly increased sIPSC frequency to 129 ± 8% of control values (n= 10, P= 0.004, Fig. 8B) without significantly depressing the late phase of sIPSC frequency (95 ± 4% of control values, n= 10, P= 0.2, Fig. 8B), suggesting that the large-conductance IK(Ca) channels are required for the effects of CCK on GABA release. Similarly, application of charybdotoxin (50 nM), another large-conductance IK(Ca) channel inhibitor, significantly increased sIPSC frequency to 162 ± 20% of control values (n= 8, P= 0.02). However, coapplication of charybdotoxin and CCK only slightly increased sIPSC frequency to 127 ± 6% of control values (n= 8, P= 0.002, data not shown), but blocked the late phase of depression (80 ± 16% of control values, n= 8, P= 0.26, data not shown). Together, these data suggest that the large-conductance IK(Ca) channels are required for the effects of CCK on GABA release.
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We next explored the signal transduction mechanisms underlying CCK-mediated modulation of GABA release. Activation of CCK-B receptors is coupled to PLC to generate two intracellular second messengers, IP3 to interact with IP3 receptors to increase intracellular Ca2+ release and diacylglycerol to activate PKC. We tested the roles of this pathway in CCK-mediated increases in GABA release. Application of the specific PLC inhibitor U 73122 (30 µM) did not block the CCK-mediated initial increase (232 ± 36% of control values, n= 8, P= 0.008, Fig. 9A) or subsequent reduction in sIPSC frequency (43 ± 15% of control values, n= 8, P= 0.01, Fig. 9A), suggesting that the activity of PLC is not required for the effects of CCK. We also examined the roles of intracellular Ca2+ release and PKC in CCK-mediated modulation of GABA release. Bath application of thapsigargin (10 µM) did not block the CCK-mediated initial increase in sIPSC frequency (212 ± 22% of control values, n= 7, P= 0.002, Fig. 9B), nor did it block the CCK-induced late phase of depression (wash for 10 min, 46 ± 7% of control values, n= 7, P= 0.0004, Fig. 9B), suggesting that intracellular Ca2+ release is not required for the effects of CCK.
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While the PLC pathway is the major intracellular pathway activated by CCK, CCK-B receptor activation is also coupled to PLA2 (Pommier et al. 1999, 2003). We next examined the effects of PLA2 on CCK-mediated modulation of GABA release. Application of the specific PLA2 inhibitor PACOCF3 (Ackermann et al. 1995; 100 µM) did not block the CCK-mediated initial increase in sIPSC frequency (CCK at 3 min, 322 ± 47% of control values, n= 5, P= 0.009, Fig. 9D) nor did it block the CCK-mediated late phase of reduction (CCK at 20 min, 53 ± 13% of control values, n= 5, P= 0.02; wash at 10 min, 35 ± 9% of control values, n= 5, P= 0.002, Fig. 9D), suggesting that the functions of PLA2 are not required for the effects of CCK on GABA release.
Since none of the inhibitors blocked the effects of CCK, we next tested a hypothesis that the effects of CCK are mediated by direct coupling of G proteins from CCK-B receptors to IK(Ca) without requiring intracellular second messengers. We used outside-out nucleated patches excised from the interneurons in the hilus to test this hypothesis. We reasoned that if intracellular second messengers are required, CCK should not change the V–I curve on outside-out patches when the integrity of intracellular signals is demolished after excision from interneurons. However, application of CCK still inhibited the outward current to 55 ± 3% of control values (at 0 mV, P < 0.001, n= 5, Fig. 9E and F), suggesting that no intracellular second messengers are required for the effects of CCK on GABA release. However, unlike the results of whole cell recordings (Fig. 7A), the outward current recorded from nucleated patches after washing in CCK-free solution was not significantly higher than the initial control values. The possible reason for the lack of over-recovery in nucleated patches is that the properties of IK(Ca) are changed after excision from the cell membrane. Nonetheless, this result further confirmed that direct coupling of G proteins from CCK-B receptors to IK(Ca) is responsible for CCK-mediated modulation of GABA release.
Developmental alteration of CCK-mediated modulation of GABA release
While most of the experiments were conducted on brain slices from 10- to 20-day-old rats because animals of this age range generate the best slices, we also expanded our experiments to include 6- and 31-day-old animals. While CCK still transiently increased, and then persistently reduced GABA release in 6-day-old animals (Fig. 10A), it had no significant effects on GABA release from 31-day-old animals (Fig. 10B), suggesting that its modulation of GABA release is developmentally regulated. We then retrospectively examined our data versus the animal ages recorded (Fig. 10C and D). The CCK-induced transient increase in sIPSC frequency was negatively correlated with the rat age (r=–0.58, P < 0.0001, Fig. 10C), but the CCK-induced late phase of reduction in GABA release was positively correlated with the animal age (r= 0.69, P < 0.0001, Fig. 10D). These results strongly indicate that CCK-mediated bidirectional modulation of GABA release is developmentally regulated and is more evident in juvenile animals.
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Ionic mechanisms underlying CCK-mediated modulation of GABA release
The magnitude and duration of the AHP are important factors in determining interspike interval and, thereby, neuronal firing rate. Our results indicate that CCK inhibits the amplitude of AHP of the interneurons in the hilus to increase action potential firing rate and GABA release. Consistent with our results, a variety of neurotransmitters (Madison & Nicoll, 1982; Pedarzani & Storm, 1995; Cloues & Sather, 2003) including neuropeptides (Jassar et al. 1999; Ogawa et al. 2005) increase action potential firing rate by depressing the AHP of action potentials. Since the AHP of the action potentials is determined by IK(Ca), we also explored the effects of CCK on IK(Ca) by using inhibitors for two types of IK(Ca) channels. Our results demonstrate that the apamin-sensitive small-conductance IK(Ca) channel does not participate in the modulation of GABA release because application of apamin alone did not change sIPSC frequency (Fig. 8A). Furthermore, the apamin-sensitive small-conductance IK(Ca) channel is unlikely to be involved in CCK-mediated modulation of GABA release because application of apamin failed to alter the effects of CCK on sIPSC frequency. However, our results demonstrate that the functions of the large-conductance IK(Ca) channel underlie CCK-mediated modulation of GABA release because the effects of CCK on sIPSC frequency were inhibited by application of the inhibitors for the large-conductance IK(Ca) channel. Consistent with our results, the large-conductance IK(Ca) channel has been reported to control the excitability of dentate gyrus (Brenner et al. 2005) and transmitter release at CA3–CA3 synapses (Raffaelli et al. 2004). More interestingly, IK(Ca) has been shown to be inhibited by a variety of neuropeptides including CCK in CA1 pyramidal neurons (Shinohara & Kawasaki, 1997), neurotensin in acutely dissociated neurons from the diagonal band of Broca (Jassar et al. 1999) and in neurons of the solitary tract nucleus (Ogawa et al. 2005) and tachykinins in NG 108-15 cells (Phenna et al. 1996). Added to this spectrum is our study showing that CCK inhibits IK(Ca) in interneurons where IK(Ca) controls spontaneous firing patterns (Goldberg & Wilson, 2005). There are two possible modes by which CCK could inhibit IK(Ca). First, CCK could inhibit Ca2+ channels to reduce Ca2+ influx, resulting in a reduction in IK(Ca). Second, CCK could directly inhibit IK(Ca) without affecting the functions of Ca2+ channels. The result that application of CCK failed to change the amplitude of Ca2+ channel currents recorded from the interneurons suggests that CCK acts by directly inhibiting IK(Ca) without modulating Ca2+ channels of the interneurons.
While our results demonstrate that the IK(Ca) channel is the major ion channel involved in CCK-mediated modulation of GABA release in the dentate gyrus region, CCK-mediated increases in GABA release in the CA1 region are related to inhibition of a resting K+ conductance (Miller et al. 1997). In fact, CCK suppresses both the resting K+ conductance and IK(Ca) in the CA1 region (Shinohara & Kawasaki, 1997). However, our results do not support a role for the inhibition of a resting K+ conductance on CCK-mediated modulation of GABA release in the dentate gyrus region because CCK had absolutely no effects on the holding current recorded from the hilar interneurons when the membrane was held at –55 mV. In fact, in both CA1 pyramidal neurons (Shinohara & Kawasaki, 1997) and hilar interneurons (Fig. 7), the major effect of CCK on the V–I curve is not within the voltages close to the resting membrane potential, but at more positive (> –40 mV) voltages, suggesting a predominantly inhibitory role on IK(Ca). Taken together, these results suggest that CCK controls GABA release by interacting with distinct ion channels in different brain regions.
In addition to modulating K+ channels, CCK has also been reported to activate cation channels in supraoptic nucleus neurons (Chakfe & Bourque, 2000, 2001) and acutely dissociated rat neostriatal neurons (Wu & Wang, 1996a, b). However, our results do not support a role for cation channels in CCK-mediated modulation of GABA release in the dentate gyrus region based on the following observations. First, CCK failed to change the holding current when the cell membrane was held at –55 mV, at which both cation channels and the K+ channels responsible for the resting membrane potentials are supposed to open. Second, application of the receptor-operated cation channel inhibitor SKF 96365 did not block the effects of CCK on sIPSCs. While our results do not support the involvement of cation channels and resting K+ channels, we cannot rule out the participation of other unidentified channels in CCK-mediated increases in GABA release because application of IK(Ca) inhibitors did not completely block CCK-mediated increases in sIPSC frequency. It is possible that CCK may have minor effects on other channels that participate in the modulation of GABA release. Further efforts are required to identify those channels.
While the mechanisms underlying the CCK-induced late phase of depression have not been completely elucidated, our results suggest that the over-recovery of IK(Ca) after CCK-induced depression is at least part of the mechanisms (Fig. 7A). The over-recovery of IK(Ca) can elevate the functions of IK(Ca) to increase the AHP, and to decrease action potential firing and GABA release. While we have observed a significant increase in IK(Ca) after washing out CCK (Fig. 7A), we failed to see statistically significant over-recovery of the AHP amplitude of action potentials (Fig. 6D), although we indeed observed that in some cells the AHP amplitudes after washing were larger than control values. The possible reason for this discrepancy is that the change in AHP amplitude is so subtle that it could not reliably be detected after averaging the action potentials. Interestingly, over-recovery of M-channel currents has also been observed after agonist-induced inhibition, and it is related to agonist-induced intracellular Ca2+ release (Marrion et al. 1991; Chen et al. 1993; Tokimasa et al. 1996; Kurennyi et al. 1997). Whether intracellular Ca2+ release underlies CCK-induced over-recovery of IK(Ca) remains to be determined.
Signalling mechanisms underlying CCK-induced modulation of GABA release
Our results demonstrate that CCK-B receptors are required for the effects of CCK on GABA release, consistent with the observations that CCK-B receptors are predominantly distributed in the hippocampus (Shigeyoshi et al. 1994) and that activation of CCK-B receptors increases GABA release from the anterior nucleus accumbens (Lanza & Makovec, 2000) and cerebral cortex (Ferraro et al. 1999; Siniscalchi et al. 2003). The next question is how activation of CCK-B receptors leads to the inhibition of IK(Ca) to increase GABA release. We first examined the roles of the second messengers coupled to CCK-B receptors, and our results do not support the involvement of any known second messengers in CCK-mediated modulation of GABA release because application of the inhibitors for PLC, intracellular Ca2+ release, PKC or PLA2 failed to block the effects of CCK on GABA release. Since G protein-coupled receptors modulate ion channels via either intracellular second messengers or direct G protein coupling, these results support a direct coupling of G proteins to CCK-B receptors and IK(Ca) channels. This notion is further supported by the result that application of CCK to the outside-out nucleated patches in which the intracellular second messengers are supposed to be disintegrated still led to an inhibition of the outward currents. Further evidence to support a direct coupling of G proteins and IK(Ca) is the time course of the effects of CCK on sIPSCs, because application of CCK increases sIPSCs frequency to the maximum in a short time (3 min), a time course too brief for second messengers to be involved. Consistent with our conclusion is the evidence showing that G proteins directly interact with IK(Ca) (Walsh et al. 1996). However, it is at present unclear whether G proteins modulate IK(Ca) via direct interaction with IK(Ca) channels or indirectly via other proteins associated with IK(Ca) channels. Further biochemical experiments are required to elucidate the interaction of G proteins and IK(Ca) channels in the interneurons.
Developmental modulation and physiological significance
We have also demonstrated that the effect of CCK on GABA release undergoes developmental modulation. The CCK-mediated initial increase and late phase of reduction are restricted to juvenile rats, but disappear when the animals are older (> 31 days). Presently, the mechanisms underlying the developmental modulation of the effects of CCK are unknown. Developmental changes in the properties of CCK-B receptors, G proteins and IK(Ca) may explain the distinct effects of CCK in animals of different ages.
There are inconsistent results regarding whether CCK modulates GABA release in different brain regions. Cholecystokinin has been reported to increase GABA release in cerebral cortex (You et al. 1997; Ferraro et al. 1999; Siniscalchi et al. 2003), neostriatum (You et al. 1996), anterior nucleus accumbens (Lanza & Makovec, 2000) and hippocampus (Perez de la Mora et al. 1993; Miller et al. 1997). By contrast, CCK has also been reported not to change GABA release in the frontal-parietal cortex (Hickling et al. 1997) and hippocampus (Migaud et al. 1994; Breukel et al. 1997). While the reasons underlying these controversies are unknown, CCK-mediated bidirectional modulation of GABA release may be relevant because most of those experiments were conducted by measuring the extracellular GABA concentration, which may reflect a neutralized effect of CCK on GABA release. Depending on the time when the samples were taken, it is likely that the CCK-mediated initial increase is attenuated by the late phase of reduction in GABA release, and the neutralized result would be no change in GABA release after application of CCK. Our results therefore provide a resolution to solve those controversies.
CCK has been implicated in modulating a variety of important brain functions including satiety, analgesia, learning and memory processes, and in neuropsychiatric disorders such as anxiety and panic attack (Sebret et al. 1999). GABAergic synaptic transmission underlies almost all of those physiological functions and neuropsychiatric disorders. For example, CCK–GABA interaction in the hippocampus has been demonstrated to elevate anxiety (Rezayat et al. 2005), which may be related to CCK-induced persistent reduction in GABA release. The present study is therefore likely to provide a novel cellular mechanism to explain the roles of CCK in these neuropsychiatric disorders and the physiological functions as well.
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