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Rapid Report |
1 Faculty of Life Sciences, University of Manchester, Manchester M60 1QD, UK
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Abstract |
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-methyl-5-HT (30 µM) mimicked the excitatory effects of serotonin. Consistently, the 5-HT2 receptor antagonist ketanserin (10 µM) fully blocked the excitatory effects of serotonin. Two prominent after-hyperpolarizations (AHPs), one of medium duration that was apamin-sensitive and followed individual spikes, and one that was slower and followed trains of spikes, were both strongly and reversibly reduced by serotonin; these effects were fully blocked by ketanserin. Conversely, the depolarizing sags observed during negative current injections and mediated by hyperpolarization-activated cationic currents were not affected. In the presence of apamin and tetrodotoxin, the slow AHP was strongly reduced by 5-HT, and fully abolished by the calcium channel blocker nickel. These results show that 5-HT exerts a powerful excitatory control on cholinergic interneurones via 5-HT2 receptors, by suppressing the AHPs associated with two distinct calcium-activated potassium currents.
(Received 14 September 2005;
accepted after revision 31 October 2005;
first published online 3 November 2005)
Corresponding author E. Bracci: Faculty of Life Sciences, University of Manchester, Manchester M60 1QD, UK. Email: e.bracci{at}manchester.ac.uk
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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) filled with a solution containing (mM): 125 potassium gluconate, 10 NaCl, 1 CaCl2, 2 MgCl2, 1 BAPTA, 19 Hepes, 0.3 guanosine triphosphate, 2 Mg-ATP, and adjusted to pH 7.3 with KOH. Gramicidin was dissolved in dimethylsulfoxide (10 mg ml–1) and then diluted in the intrapipette solution to a final concentration of 10–20 µg ml–1. The tip of the pipette was filled with gramicidin-free intracellular solution. The perforation process was considered complete when (i) the amplitude of the action potentials was steady and > 60 mV, and (ii) electrode resistance (measured with bridge compensation) was steady and < 50 M. Infrequently, an abrupt increase in the amplitude of the action potentials was observed, signalling that the membrane had ruptured, and the experiment was immediately terminated. Spike threshold was defined as the point where the temporal derivative of the membrane potential exceeded 10 mV ms–1. Values are expressed as mean ± standard deviation and statistical comparisons were made (unless otherwise stated) by Student's t test. All drugs were obtained from Tocris UK, apart from 5-HT hydrochloride, Gramicidin and NBQX, which were obtained from Sigma UK. |
Results |
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Large-size cholinergic interneurones were visually targeted and electrophysiologically identified based on: (i) the prominent medium AHP (mAHP) following individual spikes; (ii) the slow AHP (sAHP) following positive current injections which increased (or triggered) firing; and (iii) the depolarizing sag which developed during current-induced hyperpolarization (Bennett et al. 2000; Wilson, 2005; see inset of Fig. 1). Gramicidin-perforated techniques were essential to preserve the stability of these cells' properties (Wilson, 2005).
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Bath-application of serotonin (30 µM) significantly (P < 0.05) reduced the average ISI in 37/45 (82%) spontaneously active neurones. The effects of serotonin were reversible on washout. In the remaining 8/45 cells, no significant change in ISI was observed. In this subgroup of cells, subsequent application of higher concentrations of serotonin (120–240 µM) also failed to elicit a significant change in ISI. The spontaneous firing rate of these cells was not significantly different from that of the other neurones. We concluded that this subpopulation of cells was not responsive to serotonin.
When data from all 45 cells were pooled together, the average spike frequency in the presence of 30 µM serotonin was 273 ± 193% of that observed in control. Two representative examples of the action of serotonin on two cholinergic interneurones with different levels of spontaneous firing rate in control solution are shown in Fig. 1A and B. Correlation analysis showed that the effects of serotonin did not significantly depend on the level of spontaneous activity in control (correlation coefficient r=–0.13; P > 0.05), as illustrated in the raster plot of Fig. 1C.
The spontaneous activity of cholinergic interneurones and the excitatory effects of serotonin were not significantly affected by application of the ionotropic glutamate receptor antagonists nitro-7-sulfamoyl-[f]-quinoxaline-2,3-dione (NBQX; 20 µM) and D-amino-phosphonovalerate (AP5; 20 µM) (n= 5).
We tested the effects on cholinergic interneurone firing rate of different serotonin concentrations in the range 7.5–240 µM. The average effects on spike frequency for each concentration tested are illustrated in Fig. 1D. This plot was obtained by expressing the frequency observed for each concentration in a given cell as percentage of control, and then pooling together the normalized data from each cell (both responsive and non-responsive cells were included). An analysis of variance (ANOVA) test for multiple groups revealed that there was a significant dependence of the effects on serotonin concentration (P < 0.05). Further paired comparisons revealed that the effects of 7.5 µM were significantly (P < 0.05) smaller than those of 30 µM and higher concentrations, while the differences among other concentrations were not statistically significant.
The use of gramicidin-perforated patch techniques was essential to observe the effects of serotonin. Conventional whole-cell recordings revealed significant (P < 0.05) effects of 30 µM serotonin on the average ISI only in 2/12 cholinergic interneurones, in which the spike frequency was increased by < 20%.
Pharmacology of serotonin effects
In order to identify the serotonin receptors involved in these effects, we used selective ligands of 5-HT receptor classes.
The following selective agonists of postsynaptically located serotonin receptors found in the basal ganglia failed to significantly affect the average ISI of cholinergic interneurones in all cells tested: 8-OH-DPAT (5-HT1A receptor; 100 µM; n= 22); m-chlorophenyl-biguanide (5-HT3 receptor; 10 µM; n= 6); RS 67333 (5-HT4 receptor; 100 µM; n= 4); 5-carboxamidotryptamine (5-HT7 receptor; 10 µM; n= 6).
On the other hand, the excitatory effects of serotonin were mimicked by the broad spectrum 5-HT2 receptor agonist -methyl-5-HT (30 µM), as shown in the example of Fig. 2A. In 8/10 cells -methyl-5-HT significantly (P < 0.001) decreased the average ISI. On average, in the 10 cells tested, in the presence of -methyl-5-HT, spike frequency increased to 564 ± 674% of control.
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-methyl-5-HT were fully reversed by application of the 5-HT2 receptor antagonist ketanserin (10 µM), as shown in the examples of Fig. 2A and B. In a sample of five neurones from different preparations, in which serotonin significantly (P < 0.001) decreased the ISI, further application of ketanserin (in the presence of 5-HT) increased the ISI to a level which was significantly (P < 0.001) larger than those observed either in the presence of serotonin or in control solution (see plot of Fig. 2C). When applied without 5-HT, ketanserin also markedly reduced the spontaneous firing activity of cholinergic interneurones (n= 4). On average, the ISI significantly (P < 0.05) increased to 255 ± 126% of control after ketanserin application. These results showed that 5-HT2 receptors were responsible for the excitatory effects of serotonin in cholinergic interneurones, and suggested that endogenous serotonin present in the slices was enough to significantly activate 5-HT2 receptors.
Conductances modulated by 5-HT
We then investigated the membrane mechanisms modulated by 5-HT. Cholinergic interneurones display a prominent cationic current activated by hyperpolarization (Ih), which is modulated by other neurotransmitters (Pisani et al. 2003); Ih is also affected by serotonin in other brain areas (Erdemli & Crunelli, 2000). This current gives rise to slow depolarizing sags during negative current injections (Bennett et al. 2000), as apparent in the inset in Fig. 1. The amplitude and time course of the depolarizing sag (measured with 500 ms, 100–200 pA negative current injections delivered from the same membrane potential level) were not significantly affected by serotonin application (n= 6; not shown). We concluded that Ih was not modulated by 5-HT.
Another current which is prominent in cholinergic interneurones is the apamin-sensitive, calcium-activated potassium current, which gives rise to the mAHPs that follow individual spikes (Bennett et al. 2000). The amplitude of the mAHP (measured from spike threshold level to the negative peak following the spike) was on average 10.6 ± 0.5 mV. As previously reported (Bennett et al. 2000), bath-application of apamin (100 nM) strongly reduced the mAHP and induced a bursting behaviour in cholinergic interneurones (n= 4). In control solution, the mAHP was strongly and reversibly reduced by 5-HT, as shown in the example of Fig. 3A. For a better comparison of the mAHPs, in the presence of serotonin a small negative current was injected into the cells to achieve firing rates similar to control. On average, in 10 neurones selected for this analysis, the amplitude of the mAHP was significantly (P < 0.001) reduced by 29.0 ± 2.6% (Fig. 3C). Bath application of ketanserin (in the presence of 5-HT) reversed this effect, and in 6/6 cases increased the mAHP amplitude to levels significantly (P < 0.001) higher than control (Fig. 3B and C). We concluded that one of the cellular effects of 5-HT2 receptor activation is a strong reduction in the apamin-sensitive currents responsible for mAHP.
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Discussion |
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In studying these effects, it was crucial not to interfere with the intracellular milieu of the cholinergic interneurones. When performed with conventional whole-cell recordings, serotonin effects were typically absent or very weak, presumably because the metabotropic action of 5-HT2 receptors was disrupted. Gramicidin-perforated recordings allowed us to record from cholinergic interneurones for several hours without any apparent deterioration of their properties (Wilson, 2005). Under these conditions, serotonin strongly increased the spontaneous firing rate of most (> 80%) cholinergic interneurones. The remaining cells did not respond to any concentration of serotonin up to 240 µM; their firing frequency did not differ from average, suggesting that lack of serotonin effects was not due to saturation of 5-HT2 receptors by endogenous serotonin, and that they may belong to a subpopulation that does not express functional 5-HT2 receptors.
Striatal cholinergic interneurones are spontaneously active both in vivo and in vitro, thanks to a dynamic interplay of several voltage-dependent membrane conductances (Wilson, 2005) Among these, the conductances responsible for the medium and slow AHPs are particularly prominent (Bennett et al. 2000; Wilson, 2005), and play an important role in limiting the excitability of cholinergic interneurones. The mAHP sets a lower limit for the interspike interval (Stocker, 2004). The slow AHP provides a powerful negative feedback mechanism which tends to limit the duration of any excitatory input (Wilson, 2005).
The mAHP is due to SK calcium-activated potassium channels and is blocked by apamin (Bennet et al. 2000). The identity of the sAHP in cholinergic interneurones has not been fully characterized; mAHPs can be amplified by a fast, inward rectifier K+ current (KIR) sensitive to 100 µM barium (Wilson, 2005). In our experiments the sAHP observed in the presence of TTX and apamin was insensitive to barium (probably because the threshold for the auto-regenerative activation of KIR was not reached), and was completely abolished by nickel, suggesting that it was due to slow calcium-activated potassium currents similar to those observed in other regions of the brain (Stocker, 2004). Room temperature (25°C) conditions may have accentuated the serotonin effects; at this temperature, cholinergic interneurone spontaneous firing is slower than at 32°C (Bennett & Wilson, 1999), and slow AHPs are enhanced by slower removal of cytosolic calcium (Thompson et al. 1985). On the other hand, the effects of temperature on excitatory synaptic potentials (Vogalshev et al. 2000) are unlikely to have affected the present experiments, as the observed effects persisted in the presence of ionotropic glutamate receptor blockers.
Neuromodulation of the mAHP or sAHP has not been reported so far for cholinergic interneurones. In hippocampal pyramidal neurones, the sAHP has been shown to be suppressed by monoamine neurotransmitters through activation of protein kinase A (Stocker, 2004) and by muscarinic receptor activation (Egorov et al. 1999). Interestingly, 5-HT4 and 5-HT7 receptor activation reduces the sAHP in hippocampal neurones (Torres et al. 1994; Bacon & Beck, 2000). There is evidence that the action of neurotransmitters on the sAHP in hippocampal neurones is exerted directly on the underlying calcium-activated potassium conductances, rather than on calcium channels (Sah & Faber, 2002). In motoneurones, the mAHP is reduced by serotonin through 5-HT1A receptor-mediated inhibition of calcium channels (Bayliss et al. 1995). On the other hand, recent evidence has shown that 5-HT1A receptor activation can also directly affect SK channels that underlie the mAHP (Grunnet et al. 2004). A direct action of serotonin on calcium-dependent potassium conductances, is also consistent with the observation that G-protein-coupled 5-HT2 receptors increase the hydrolysis of inositol phosphates and elevate cytosolic calcium (Hoyer et al. 2002).
Further experiments will be required to determine whether, in cholinergic interneurones, serotonin directly inhibits calcium-dependent potassium channels, or whether its action is due to suppression of calcium channels. Producing evidence will pose serious challenges; calcium-dependent potassium channels are functionally coupled to different subtypes of voltage-activated calcium channels (including L-, N-, P-, R- and T-types) in different neuronal types (Stocker, 2004), and recent work by Goldberg and Wilson (2005) has shown that in striatal cholinergic interneurons Cav2.2 (N-type) calcium channels are functionally associated with the mAHP, while Cav1 (L-type) are selectively associated with the sAHP. Furthermore, studying calcium channel modulation by serotonin will require voltage-clamp experiments, which are difficult to perform with gramicidin-perforated patch techniques, due to the high access resistance of the electrode (Wilson, 2005). High-resolution calcium imaging will perhaps be required to resolve this issue. The absence of effects on membrane potential in the presence of TTX suggests that leak conductances were not modulated by serotonin.
Our pharmacological experiments straightforwardly identified 5-HT2 receptors as responsible for serotonin effects. These G-coupled receptors are abundant in the striatum (Ward & Dorsa, 1996) and modulate neurones in other brain areas, although their ability to affect the AHPs has not been reported so far. The observation that the 5-HT2 receptor antagonist ketanserin (when applied without 5-HT) reduced the spontaneous firing of cholinergic interneurones to levels lower than those observed in control solution suggests that endogenous serotonin in the slice significantly activated 5-HT2 receptors, and this activation was abolished by ketanserin. Alternatively, ketanserin may have acted as an inverse agonist, shifting the balance of 5-HT2 receptors towards the non-active conformational state (Dupre et al. 2004).
Cholinergic interneurones fire tonically in vivo, and changes in their firing activity encode behaviourally relevant information (Yamada et al. 2004; Morris et al. 2004). In the striatum, a host of pre- and postsynaptic membrane mechanisms are controlled by acetylcholine. These membrane mechanisms will undergo phasic changes according to the variations in firing rate of local cholinergic interneurones. The ability of serotonin to strongly increase this firing suggests that the operation of the striatal networks is profoundly affected by serotonin release. Thus, the present data provide a novel cellular explanation for the serotoninergic control of the striatum, which may be relevant for the development of new rational therapies for motor and psychiatric illnesses.
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