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-Adrenoceptive Dual Modulation of Inhibitory GABAergic Inputs to Purkinje Cells in the Mouse Cerebellum
Neuronal Circuit Mechanisms Research Group, Brain Science Institute, RIKEN, Saitama, Japan
Submitted 6 June 2005; accepted in final form 20 October 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-adrenoceptor–and cyclic AMP–dependent pathways. However, the effects of 
-adrenoceptor activation on cerebellar inhibitory neurotransmission have not yet been fully elucidated. Therefore we investigated the effects of the 1- or 2-adrenoceptor agonist on inhibitory postsynaptic currents (IPSCs) recorded from mouse Purkinje cells (PCs). We found that the nonselective -adrenoceptor agonist 6-fluoro-norepinephrine increased both the frequency and amplitude of spontaneous IPSCs (sIPSCs). This enhancement was mostly mimicked by the selective 1-adrenoceptor agonist phenylephrine (PE). PE also enhanced the amplitude of evoked IPSCs (eIPSCs) and increased the frequency but not the amplitude of miniature IPSCs (mIPSCs). Moreover, PE decreased the paired-pulse ratio of eIPSCs and did not change 
-aminobutyric acid (GABA) receptor sensitivity in PCs. Conversely, the selective 2-adrenoceptor agonist clonidine significantly reduced both the frequency and the amplitude of sIPSCs. Neither eIPSCs nor mIPSCs were affected by clonidine. Furthermore, presynaptic cell-attached recordings showed that spontaneous activity of GABAergic interneurons was enhanced by PE but reduced by clonidine. These results suggest that NA enhances inhibitory neurotransmitter release by 1-adrenoceptors, which are expressed in presynaptic terminals and somatodendritic domains, whereas NA suppresses the excitability of interneurons by 2-adrenoceptors, which are expressed in presynaptic somatodendritic domains. Thus cerebellar -adrenoceptors play roles in a presynaptic dual modulation of GABAergic inputs from interneurons to PCs, thereby providing a likely mechanism for the fine-tuning of information flow in the cerebellar cortex. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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- and -adrenoceptors to serve as a neurotransmitter for modulating the synaptic strength in widespread brain areas. Among them is the cerebellar cortex where NA-containing afferent fibers originated from the locus coeruleus in the brain stem display a diffuse pattern of projections like those in other brain areas (Bloom et al. 1971
; Hökfelt and Fuxe 1969). The stimulation of the locus coeruleus has been shown to elicit a prolonged inhibition of the electrical activity of Purkinje cells (PCs) and this effect is blocked by adrenoceptor antagonists (Hoffer et al. 1973; Woodward et al. 1979). Moreover, several studies suggest that NA modulates cerebellum-dependent learning tasks, mostly through activation of -adrenoceptors (for a review see Cartford et al. 2004). It has also recently been demonstrated that NA enhances cerebellar GABAergic transmission onto interneurons and PCs by -adrenoceptor–dependent pathways (Cheun and Yeh 1996; Kondo and Marty 1997, 1998; Llano and Gerscenfeld 1993b; Mitoma and Konishi 1999; Saitow et al. 2000). Morphological studies have shown that not only - but also -adrenoceptors are present in the cerebellar cortex: for example, in situ hybridization studies indicate that 1A mRNA among the three subtypes of 1 (1A, 1B, and 1D) is moderately expressed in the cerebellum (Docherty 1998), whereas mRNAs of the other two subtypes are scarcely detected (Day et al. 1997). Based on their pharmacological properties and molecular structure, 2-adrenoceptors have been subdivided into 2A, 2B, and 2C subtypes (Docherty 1998). PCs in the cerebellum express 2B-adrenoceptor mRNA (Wang et al. 2002; Winzer-Serhan and Leslie 1997). 2C-Adrenoceptor mRNA has been shown to be transiently expressed in the internal granular layer and the molecular layer during the critical period of granule cell development (Winzer-Serhan et al. 1997).
In contrast to -adrenoceptors, relatively little is known about physiological roles of -adrenoceptors in the cerebellar cortex. It has been reported that 1-adrenoceptor activation increases intracellular Ca2+ concentration ([Ca2+]i) in PCs and Bergmann glia by phosphatidylinositol (PI) turnover and indirectly modulates neurotransmission, presumably depending on liberation of retrograde messengers (Kirischuk et al. 1996a,b; Kulik et al. 1999). However, the underlying mechanisms of -adrenoceptor–mediated regulation of cerebellar synaptic transmission remain elusive.
In other brain areas, several studies have reported that the activation of 1-adrenoceptors modifies inhibitory synaptic transmission. NA suppresses the amplitude of evoked inhibitory postsynaptic potentials (eIPSPs) with an increment of the spontaneous IPSP frequency at hippocampal synapses (Madison and Nicoll 1988). It is also reported that NA increases through 1-adrenoceptors not only the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but also the rate of action potential firing in hippocampal interneurons without influencing miniature IPSCs (mIPSCs) (Bergles et al. 1996). Similar 1-adrenoceptor–mediated increments of the sIPSC frequency have been reported in the frontal cortex (Kawaguchi and Shindou 1998), the sensory motor cortex (Bennett et al. 1998), and the hypothalamic paraventricular nucleus (Han et al. 2002). On the contrary, 2-adrenoceptor activation has been reported to decrease the frequency of sIPSCs in the hypothalamic paraventricular nucleus (Han et al. 2002; Li et al. 2005). Therefore it is conceivable that the activation of both 1- and 2-adrenoceptors in the cerebellum differentially modulates inhibitory GABAergic transmission. Previous studies reported that the bath application of the selective 1-adrenoceptor agonist phenylephrine (PE) or the selective 2-adrenoceptor agonist clonidine affects neither the frequency nor amplitude of IPSCs in PCs and stellate cells (Llano and Gerschenfeld 1993b; Mitoma and Konishi 1999). Furthermore, Kondo and Marty (1998) have shown that the application of the -adrenoceptor agonist 6-fluoro-norepinephrine (6FNE), which activates both 1- and 2-adrenoceptors, induces either a small increase or decrease in the firing rate of individual stellate cells in the rat cerebellar cortex. However, the precise effects of the differential activations of -adrenoceptors in the cerebellum have yet to be determined. Using selective adrenoceptor agonists, this study therefore aimed at determining the effects of 1- or 2-adrenoceptor activation on GABAergic transmission between interneurons and PCs in the mouse cerebellar cortex. We found that 1-adrenoceptor activation enhanced IPSCs through not only regulation of neurotransmitter release in presynaptic terminals but also facilitation of spontaneous activity in presynaptic somatodendritic domains, whereas 2-adrenoceptor activation reduced sIPSCs resulting from suppression of presynaptic firing in presynaptic somatodendritic domains. These results suggest that -adrenoceptors cause a dual modulation of inhibitory synaptic transmission at interneuron–PC synapses in the cerebellar cortex. This dual modulation could participate in fine-tuning for the firing of PCs, the sole output from the cerebellar cortex, in synergy with -adrenoceptors.
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METHODS |
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C57BL/6 mice (PND 18–25) were anesthetized with diethyl ether, and sagittal slices (220 µm thick) of cerebellar vermis were prepared using a vibrating microtome (VT1000S, Leica, Nussloch, Germany). Whole cell recordings were obtained from PCs visually identified under Nomarski optics using a water-immersion objective (63x, NA 0.90, Olympus, Tokyo, Japan) as described previously (Hirono et al. 2001). The slices were superfused with artificial cerebrospinal fluid (ACSF) containing (in mM) 138.6 NaCl, 3.35 KCl, 21 NaHCO3, 0.6 NaH2PO4, 9.9 glucose, 2 CaCl2, and 1 MgCl2, and gassed with a mixture of 95% O2-5% CO2 (pH 7.4). Patch pipettes (2–3 M
) were filled with an intracellular solution containing (in mM) 140 KCH3SO3, 5 KCl, 0.1 CaCl2 1.0 K-EGTA, 10.0 Na-HEPES, 3.0 Mg-ATP, and 0.4 Na-GTP (pH 7.4). The holding potential was set at –55 to –45 mV. We used a potassium methanesulfonate–based internal solution to record the excitatory synaptic current as an inward current, which is mediated by ionotropic glutamate receptors, and the inhibitory synaptic current as an outward current, which is mediated by ionotropic GABAergic receptors. To specifically isolate inhibitory outward currents from excitatory inward currents, a nonselective glutamate ion channel inhibitor, kynurenic acid (1 mM), was added to the ACSF throughout the recordings. IPSCs were completely abolished by bicuculline (10 µM). mIPSCs were recorded using a CsCl-based internal solution containing (in mM) 140 CsCl, 0.1 CaCl2, 1.0 K-EGTA, 10.0 Na-HEPES, 3.0 Mg-ATP, and 0.4 Na-GTP (pH 7.4), instead of the potassium methanesulfonate–based internal solution, in the presence of tetrodotoxin (TTX, 0.2 µM) and kynurenic acid (1 mM) from PCs voltage-clamped at –70 to –65 mV. For presynaptic loose cell-attached recordings, the pipette was gently placed in contact with an interneuron located in the external half of the molecular layer and slight suction was applied. The pipette containing ACSF was maintained at 0 mV. According to the location of the interneurons, we recorded extracellular firing mainly from stellate cells. However, because we did not identify each interneuron as either a basket or stellate cell by using previously reported morphological and physiological criteria (Häusser and Clark 1997; Llano and Gerschenfeld 1993a), we generically refer to recorded cells as interneurons. Membrane currents were recorded using an Axopatch 700B amplifier (Axon Instruments, Foster City, CA) and pCLAMP8 software (Axon Instruments), digitized, and stored on a computer disk for off-line analysis. All signals were filtered at 2 kHz and sampled at 5–10 kHz, and sIPSCs and mIPSCs were analyzed with a threshold of 10 pA. Focal stimulation (30–50 V, 0.1–0.2 ms) was applied by a glass microelectrode containing ACSF placed within the molecular layer in the cerebellar cortex. Series resistance (8–14 M) was monitored using a –5-mV hyperpolarizing voltage pulse (30–50 ms) every 30 s, and experimental data were discarded if the value changed by >20%. -Aminobutyric acid (GABA, 100 mM) was applied by iontophoresis through microelectrodes placed in the vicinity of the proximal dendrites of PCs from which recordings were obtained. All experiments were performed at room temperature (23–26°C).
Drugs
RS79948, (RS)--methyl-4-carboxyphenylglycine [(RS)-MCPG], (RS)--cyclopropyl-4-phosphonophenylglycine (CPPG), and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were obtained from Tocris Cookson (Bristol, UK); tetrodotoxin (TTX) was obtained from Wako (Osaka, Japan). All other chemicals were from Sigma (St. Louis, MO).
Data analysis and statistics
sIPSCs, mIPSCs, and action potential frequencies were analyzed using the Mini analysis program, version 6 (Synaptosoft, Decature, GA) and Kyplot software (Kyence, Tokyo, Japan). All data are expressed as mean ± SE. Unless otherwise stated, the level of significance was determined by paired Student's t-test between groups.
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RESULTS |
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-adrenoceptor agonist on sIPSCs
In this study, we recorded sIPSCs as outward current responses from mouse cerebellar PCs voltage-clamped at –55 to –45 mV. We first investigated the effects of the bath application of the nonselective -adrenoceptor agonist 6FNE on sIPSCs. In 18 of 21 PCs recorded, 6FNE (30 µM) increased the frequency of sIPSCs (Fig. 1, A–C), whereas in the remaining three PCs 6FNE reduced the sIPSC frequency (Fig. 1C). The mean frequency for the sIPSCs changed from 13.4 ± 0.9 to 16.7 ± 1.2 Hz (127 ± 6% of control, ranging from 87 to 191%; n = 21; P < 0.001). The 6FNE-induced enhancement subsided over 10 min after removing the agonist (Fig. 1B). 6FNE also prominently increased the amplitude of sIPSCs (P < 0.05, by Kolmogorov–Smirnov test; Fig. 1D). The mean amplitude of sIPSCs changed from 29.4 ± 2.2 to 33.3 ± 2.9 pA (113 ± 2% of control; n = 21; P < 0.001). These results are in a striking contrast to previous studies showing that -adrenoceptor agonists do not induce any systematic change in the IPSCs recorded from stellate cells and PCs (Llano and Gershenfeld 1993b; Mitoma and Konishi 1999; but see Kondo and Marty 1998). The diverse distribution of the -adrenoceptor effects on the sIPSC frequency (Fig. 1C) may reflect combinations of opposite effects of 1- and 2-adrenoceptor activation at cerebellar inhibitory synapses. To confirm the involvement of 1-adrenoceptors in the 6FNE-evoked effects, we examined the effects of the selective 1-adrenoceptor antagonist prazosin (10 µM, pretreatment for 15 min) on the 6FNE-induced responses. Prazosin reduced the sIPSC frequency by 13 ± 1% of the control (P < 0.01) 15 min after its administration, likely suggesting that basal level of sIPSCs could be affected by background activity of 1-adrenoceptors, and completely abolished the 6FNE-mediated increase in the sIPSC frequency (104 ± 2% of control; n = 5; P > 0.1; Fig. 1E) and the sIPSC amplitude (107 ± 3% of control; n = 5; P > 0.1). In one of five PCs tested, 6FNE decreased the sIPSC frequency in the presence of prazosin. These results were confirmed using another selective 1-adrenoceptor antagonist, corynanthine (10 µM) (data not shown). On the other hand, in the presence of the selective 2-adrenoceptor antagonist yohimbine (10 µM), 6FNE had a tendency to increase the sIPSC frequency more than in the absence of the antagonist (142 ± 4% of control, ranging from 134 to 152%; n = 4; P < 0.05). These results suggest that 6FNE mainly enhances sIPSCs by 1-adrenoceptor activation, whereas this agonist may produce an opposite effect in some PCs by 2-adrenoceptor activation.
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1-adrenoceptor activation facilitates IPSCs
To elucidate the effects of 1-adrenoceptor activation on IPSCs at interneuron–PC synapses, we applied the selective 1-adrenoceptor agonist PE to the mouse cerebellar slices. Exogenously applied PE (30 µM) increased the frequency of sIPSC from 13.3 ± 0.7 to 16.8 ± 0.6 Hz (129 ± 4% of control, ranging from 111 to 169%; n = 16; P < 0.001; Fig. 2, A–D). This PE-mediated facilitation of the sIPSC frequency was dose dependent with a half-maximal effective concentration (EC50) of 1.91 µM (Fig. 2E). PE also significantly increased the amplitude of sIPSCs as shown by the cumulative probability curves (P < 0.001, by Kolmogorov–Smirnov test; Fig. 2F). The mean amplitude of sIPSCs changed from 24.8 ± 1.4 to 30.5 ± 1.6 pA (124 ± 3% of control; n = 16; P < 0.001).
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1-adrenoceptor activation elicits the IPSC potentiation by a presynaptic or postsynaptic mechanism. First, we compared the paired-pulse (PP) ratios before and during PE administration. PE decreased the PP ratio as shown in Fig. 3D (from 1.22 ± 0.10 to 1.09 ± 0.08; n = 10; P < 0.01). To further explore whether postsynaptic GABA receptor responses are influenced by 1-adrenoceptor activation, we applied GABA iontophoretically to PCs and examined GABA current responses in the presence or absence of PE (30 µM). Although PE potentiated sIPSCs, it had little effect on the GABA current responses (104 ± 3% of control; n = 3; P > 0.3; Fig. 3E), indicating that PE does not change GABA receptor sensitivity. These results strongly suggest that the 1-adrenoceptor–mediated IPSC potentiation involved an enhancement of the release probability at the presynaptic terminals (Zucker and Regehr 2002).
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1-adrenoceptor–mediated IPSC potentiation is mediated by a presynaptic GABA release enhancement.
Retrograde messengers do not contribute to 1-adrenoceptor–mediated IPSC potentiation
It is possible that activation of 1-adrenoceptors in PCs and Bergmann glia could induce the release of retrograde messengers, resulting in the facilitation of GABA release from cerebellar interneurons. Agulló et al. (1995) reported that the 1-adrenoceptor activation in glial cells facilitates nitric oxide (NO) synthase. To test these possibilities, we examined the effects of antagonists of possible retrograde messengers (i.e., glutamate, ATP, NO, and endocannabinoid) on the PE-evoked sIPSC potentiation. In our experiments we used kynurenic acid as a broad-spectrum antagonist of all ionotropic GluRs to isolate IPSCs (see above). We applied the mGluR antagonists, MCPG for groups I and II, and CPPG for group III, and examined the PE-induced facilitation of sIPSCs. In the presence of MCPG (300 µM) or CPPG (300 µM), PE (30 µM) significantly increased the sIPSC frequency (MCPG; 131 ± 4% of control; n = 4; P < 0.01, CPPG; 133 ± 7% of control; n = 3; P < 0.05) and the sIPSC amplitude (MCPG; 126 ± 3% of control; n = 4; P < 0.05, CPPG; 152 ± 17% of control; n = 3; P < 0.05). Furthermore, in the presence of a P2 purinoceptor antagonist, PPADS (10 µM), the PE-mediated increase in the sIPSC frequency was observed (125 ± 4% of control; n = 6; P < 0.01). The mean magnitude of the PE-induced enhancement of the sIPSC amplitude with PPADS was 147 ± 13% of control (n = 6; P < 0.05). The cerebellar slices were pretreated for 5 min with a NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 µM) and then PE (30 µM) was applied. The PE-mediated increase in the sIPSC frequency was similarly observed (133 ± 7% of control; n = 4; P < 0.05). Therefore the PE-induced enhancement of sIPSCs in the presence of all of these antagonists was not different from that without these antagonists, suggesting that PE directly acts on 1-adrenoceptors in presynaptic GABAergic interneurons and causes the facilitation of GABAergic transmitter release.
Finally, we examined the counteracting effect of endocannabinoid on the 1-adrenoceptor–induced sIPSC potentiation. We administrated a CB1R antagonist, AM251 (0.5 µM), which caused a slight change in the basal sIPSC frequency in PCs (99 ± 8% of control; n = 3; P > 0.8), and did not have any effects on the magnitude of the PE-mediated potentiation of the sIPSC frequency (126 ± 7% of control; n = 3; P < 0.05) or the sIPSC amplitude (139 ± 12% of control; n = 3; P < 0.05).
Activation of 2-adrenoceptors attenuates IPSCs
We further tested the effects of the 2-adrenoceptor agonist clonidine on sIPSCs recorded from PCs. The bath application of clonidine (30 µM) induced a significant decrease in the sIPSC frequency from 13.5 ± 1.0 to 10.4 ± 1.0 Hz (74 ± 3% of control; n = 17; P < 0.001; Fig. 5, A–D) and significantly reduced the sIPSC amplitude (P < 0.01, by Kolmogorov–Smirnov test; Fig. 5E). The mean sIPSC amplitude changed from 26.2 ± 1.6 to 23.1 ± 1.5 pA (89 ± 2% of control; n = 17; P < 0.001). The sIPSC frequency returned to the original level within several minutes after clonidine washout. Clonidine at a higher concentration (100 µM) did not increase the reduction rate of the sIPSC frequency compared with 30 µM clonidine (76 ± 4% of control; n = 9). To determine the specific effect of clonidine, we used selective 2-aderenoceptor antagonists, RS79948 and yohimbine. The above-mentioned effect of clonidine on the sIPSC frequency was completely blocked by a 10-min pretreatment with RS79948 (10 µM) (95 ± 3% of control; n = 5; P > 0.1) or yohimbine (10 µM) (105 ± 13% of control; n = 3; P > 0.9) (Fig. 5D). Additionally, we examined the effects of 2-adrenoceptor activation on eIPSCs in PCs. Clonidine did not change the amplitude of eIPSCs (97 ± 1% of control; n = 6; P > 0.06) or the PP ratio (101 ± 4% of control; n = 6; P > 0.9; Fig. 6, A–C). To test whether postsynaptic GABA responses are affected by 2-adrenoceptor activation, we applied GABA iontophoretically to PCs and examined GABA current responses before and during clonidine (30 µM) administration. Although clonidine attenuated sIPSCs, it had no effect on the peak amplitude of GABA-induced current responses (96 ± 3% of control; n = 3; P > 0.3), indicating that 2-adrenoceptors do not change GABA receptor sensitivity or conductance at postsynaptic sites. Therefore 2-adrenoceptors attenuate inhibitory synaptic transmission, which is mainly mediated through a presynaptic mechanism.
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2-adrenoceptors in the presynaptic interneurons could suppress action potential generation.
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DISCUSSION |
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-adrenoceptor activation elicits a bidirectional effect on both the frequency and amplitude of sIPSCs at interneuron–PC synapses in the mouse cerebellar cortex. 1-Adrenoceptor activation facilitates sIPSCs through the enhancement of action potential generation in presynaptic GABAergic interneurons and presumably by [Ca2+]i elevation in presynaptic terminals. In contrast, 2-adrenoceptor activation decreases sIPSCs by suppressing spontaneous firing activity in presynaptic interneurons.
Presynaptic potentiation of IPSCs by 1-adrenoceptor activation
In this study, the selective 1-adrenoceptor agonist PE increased the sIPSC frequency and amplitude, and the eIPSC amplitude with a reduction in the PP ratio in mouse cerebellar PCs. Additionally, the bath application of PE did not affect postsynaptic GABA receptor sensitivity or the mIPSC amplitude except for increasing the mIPSC frequency. Furthermore, the activation of 1-adrenoceptors in presynaptic interneurons enhanced spontaneous activity. These observations indicate that the 1-adrenoceptor–induced potentiation of IPSCs involves presynaptic mechanisms underlying an enhancement of the release probability of GABA at presynaptic terminals as well as the facilitation of action potential induction most likely in somatodendritic domains. In this study, we did not explore the specific biochemical mechanism underlying the 1-adrenoceptor–mediated IPSC potentiation; however, the mechanism likely involves an increase in [Ca2+]i because the 1-adrenoceptor is coupled to G-protein Gq, which facilitates Ca2+ release from Ca2+ stores through PI turnover stimulation (Docherty 1998; Kirischuk et al. 1996a). Additionally, it is known that the activation of 1-adrenoceptor elicits protein kinase C (PKC) activation (Docherty 1998), and that PKC activation phosphorylates presynaptic proteins involved in vesicle fusion, leading to the modulation of synaptic transmitter release (Leenders and Sheng 2005). It has been reported that phorbol 12,13-dibutyrate (PDBu), an activator of PKC, increases the frequency of mIPSCs in mouse PCs (Harvey and Stephens 2004). We also observed that the application of PDBu (2 µM) increased the sIPSC frequency (131 ± 3% of control; n = 4; P < 0.05), moreover, in the presence of PDBu, PE (30 µM) made a smaller increase in the sIPSC frequency (112 ± 3% of control; n = 4) than that in the absence of PDBu (P < 0.05). Therefore it can be assumed that 1-adrenoceptor activation in presynaptic terminals elicits not only [Ca2+]i elevation as previously reported in PCs and Bergmann glia (Kirischuk et al. 1996a,b; Kulik et al. 1999), but also PKC activation, leading to the facilitation of inhibitory transmitter release onto PCs.
There is a possible alternate mechanism underlying the 1-adrenoceptor–mediated potentiation of IPSCs, in which retrograde messengers from PCs and Bergmann glia participate. It is considered that [Ca2+]i elevation by 1-adrenoceptor activation in PCs can evoke the release of glutamate, endocannabinoid, and other unknown substances. Retrograde glutamate enhances GABA release at cerebellar interneuron–PC synapses by activating presynaptic N-methyl-D-aspartate (NMDA) receptors (Duguid and Smart 2004; Glitsch and Marty 1999; Huang and Bordey 2004) or group I mGluRs (Karakossian and Otis 2004). However, because we applied kynurenic acid in our experiments, and mGluR antagonists did not inhibit the PE-induced sIPSC potentiation, we ruled out the possible involvement of presynaptic glutamate receptors in the 1-adrenoceptor–mediated IPSC potentiation. It is likely that endocannabinoid can partially mask the PE-induced potentiation of sIPSCs because endocannabinoid suppresses sIPSCs in the cerebellum (Diana et al. 2002; Galante and Diana 2004; Kreitzer and Regehr 2001; Yoshida et al. 2002). The percentage of PE-induced potentiation did not change in the presence or absence of CB1R antagonist AM-251, suggesting that the 1-adrenoceptor–induced [Ca2+]i elevation in PCs is not sufficiently effective to induce endocannabinoid-mediated synaptic modulation. This result could be explained by two possibilities. First, the magnitude of [Ca2+]i elevation through 1-adrenoceptor activation is not sufficient to release endocannabinoid from PCs. Second, postsynaptic domains, wherein 1-adrenoceptor–induced [Ca2+]i elevation occurs and endocannabinoid is isolated, could be far from the GABAergic inhibitory synaptic sites.
Furthermore, Bergmann glia can release ATP and glutamate through 1-aderenoceptor activation, and then ATP activates purinoceptors in presynaptic interneurons to facilitate sIPSCs (Brockhaus et al. 2004; Saitow et al. 2005). To elucidate the participation of ATP action in the 1-adrenoceptor–mediated IPSC enhancement, we perfused a P2 purinoceptor antagonist, PPADS. The percentage of PE-induced sIPSC enhancement did not change in the presence of PPADS, indicating that the 1-aderenoceptor–induced potentiation is not mediated by P2 purinoceptor activation in presynaptic neurons. This result was supported by the discrepancy in the effects on mIPSCs between the 1-aderenoceptor and the P2 purinoceptor. The frequency of mIPSCs is enhanced by the activation of 1-adrenoceptors but not P2 purinoceptors at interneuron–PC synapses (Brockhaus et al. 2004).
Presynaptic suppression of IPSCs by 2-adrenoceptor activation
This study shows that presynaptic 2-adrenoceptors mediate the suppression of GABA release in the cerebellar cortex. 2-Adrenoceptor activation by clonidine did not elicit any change in the PP ratio or the mIPSC frequency. Moreover, clonidine did not affect GABA receptor sensitivity or the mIPSC amplitude. These findings suggest that 2-adrenoceptors suppress GABA release by attenuating spike generation presumably at somatodendritic domains. This can be supported by the result that the activation of 2-adrenoceptors reduced the firing rate of presynaptic interneurons. The 2-adrenoceptor is most commonly linked to Gi/o proteins, whose activation leads to the reduction in intracellular cAMP concentration, inhibition of voltage-gated Ca2+ channels, and activation of inwardly rectifying K+ channels (Bylund 1995). These phenomena seem to contribute to the decrease in the firing rate of presynaptic GABAergic neurons and/or in the number of functional presynaptic release sites.
Possible explanations for discrepancy in results from previous reports
Our results differ from those of previous studies, which showed that 6FNE (30 µM) had no effect on IPSCs in rat cerebellar PCs (Mitoma and Konishi 1999), and that the bath applications of PE (10 µM) and clonidine (5 µM) affected neither the IPSC frequency nor amplitude in rat stellate cells (Llano and Gerschenfeld 1993b). However, a previous report showing that 6FNE induces either a small increase or decrease in the firing frequency of rat cerebellar stellate cells (Kondo and Marty 1998) intimates our observations. Because previous studies used rats, whereas we used mice, the discrepancy in the -adrenoceptor–mediated effects could have arisen from differences between rodent species. The mouse cerebellum contains the greatest density of tyrosine hydroxylase (TH)–labeled fibers when compared with the opossum and the cat cerebellum (Nelson et al. 1997). The expression level of presynaptic -adrenoceptors may be higher in the mouse cerebellum than that in the rat cerebellum. If so, synaptic transmission in the mouse cerebellum is modulated by NA through various types of adrenoceptors compared with other rodents, and it might be difficult to detect the effects of -adrenoceptors on sIPSCs in the rat cerebellum. Another possible reason is that most previous studies used only one concentration of agonists, which was slightly lower than that applied here, although we observed a substantial increase in the sIPSC frequency by PE at under micromolar concentrations. Moreover, in the previous studies, sIPSCs were recorded in the cerebellum of young rats at the age from PND 12 (or 14) (Kondo and Marty 1998; Llano and Gerschenfeld 1993b; Mitoma and Konishi 1999), whereas we used mice at PND 18–25. Therefore there is a possibility that the -adrenoceptor effects on sIPSCs can developmentally change.
Functional implication of presynaptic 1-, 2-, and 2-adrenoceptor activation
The inhibitory inputs to PCs regulate the excitability of PCs (Häusser and Clark 1997), and they can be activated by presynaptic -adrenoceptors when NA is released from cerebellar noradrenergic fibers (Cheun and Yen 1996; Kondo and Marty 1997, 1998; Llano and Gerscenfeld 1993b; Mitoma and Konishi 1999; Saitow et al. 2000). The -adrenoceptor agonist isoproterenol increases the mIPSC frequency (Mitoma and Konishi 1999) and the isoproterenol-mediated enhancement of IPSCs is blocked by the selective 2-adrenoceptor antagonist ICI118,551 (Saitow et al. 2000). On the other hand, some studies have shown that 1-adrenoceptor activation inhibits PC activity, as we describe here, thus disinhibiting the deep cerebellar nuclei (Crepel et al. 1987; Parfitt et al. 1988). Therefore it is possible that 1- and 2-adrenoceptors are colocalized at the inhibitory presynaptic terminals, where NA facilitates GABA release by activation of both 1- and 2-adrenoceptors.
We also observed a PE-mediated increase and a clonidine-mediated decrease in the rate of action potentials in interneurons. The present results demonstrate that 1-adrenoceptors, which are expressed not only in presynaptic terminals but also most likely in somatodendritic membranes, increase the rate of action potential, whereas 2-adrenoceptors, which are presumably expressed in presynaptic somatodendritic domains, suppress action potential generation in interneurons. It is very important to understand the physiological roles of the two -adrenoceptors' opposite effects in inhibitory synaptic modulation in the cerebellar cortex. One plausible role is that when NA diffuses into the cerebellar cortex, 2-adrenoceptor activation appears to prevent the overexcitation of presynaptic interneurons induced by 1- and 2-adrenoceptor activation. We propose that NA regulates cerebellar signal processing in the balance of activation of all adrenoceptor subtypes expressed in presynaptic interneurons.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Hirono, Neuronal Circuit Mechanisms Research Group, Brain Science Institute, RIKEN 2-1 Hirosawa, Wako, Saitama 351-0198, Japan (E-mail: hironom{at}brain.riken.jp)
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