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Serotonin depolarizes hippocampal interneurones in the rat stratum oriens by interaction with 5HT2 receptors
Neuroscience Letters 270 (1999) 56±58
Serotonin depolarizes hippocampal interneurones in the rat stratum oriens by interaction with 5HT2 receptors K. Lee*, A.K. Dixon, R.D. Pinnock Parke-Davis Neuroscience Research Centre, Cambridge University Forvie Site, Robinson Way, Cambridge, CB2 2QB, UK Received 20 February 1999; received in revised form 14 May 1999; accepted 21 May 1999
Abstract Patch-clamp recording techniques were used to examine the effect of serotonin (5HT) upon interneurones contained in the stratum oriens layer of hippocampal slices. Bath application of 1±20 mM 5HT depolarized neurones by the induction of an inward current at 260 mV. This inward current was Na 1-dependent in nature, was mimicked by the 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and was inhibited by pre-incubation with the 5HT2 receptor antagonist ritanserin. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Whole-cell patch clamp; Serotonin; Hippocampus
The mammalian hippocampus receives a dense serotonergic innervation from the raphe nuclei and incoming ®bres make synaptic contacts on both pyramidal neurones and inhibitory GABAergic interneurones [1,8]. Although electrophysiological studies have revealed a variety of serotonin (5HT)-mediated responses in pyramidal neurones [2,11] much less is known about the effects of this neurotransmitter on inhibitory interneurones. Previous studies have shown that 5HT causes a large increase in spontaneous GABAergic synaptic potentials in hippocampal CA1 pyramidal neurones [5,10]. This effect is thought to arise at least in part through 5HT3 mediated depolarization of interneurones in the stratum lacunosum [7]. However, Shen and Andrade [12] have recently provided evidence to suggest that 5HT2 receptor stimulation also enhances GABAergic transmission in the hippocampus. In the present study we demonstrate that these effects may arise from interneurones situated within the stratum oriens layer. Brains were removed from 14±28 day-old male Wistar rats and 300 mm coronal slices containing the hippocampus were prepared in physiological saline using a vibratome. In all experiments neurones were visualized using infrared differential interference contrast videomicroscopy [13]. The physiological saline contained (mM) 125.0 NaCl, 25.0 NaHCO3, 10.0 glucose, 2.5 KCl, 1.25 NaH2PO4, 2.0 * Corresponding author. Tel.: 144-1223-210-929; fax: 1441223-249-106. E-mail address: [email protected] (K. Lee)
CaCl2, 1.0 MgCl2 and was bubbled with a 95%, 5% O2/CO2 gas mixture. This solution was also used as the extracellular solution in all experiments. The intracellular (pipette) solution comprised (mM) 120.0 Kgluconate, 10.0 NaCl, 2.0 MgCl2, 1.0 K2EGTA, 10.0 HEPES, 4.0 Na2ATP, 0.3 Na2GTP, pH adjusted to 7.2 with KOH. All experiments were conducted at 30±358C. Electrophysiological recordings were performed as previously described using borosilicate glass recording electrodes with resistances of 3±6 MV when ®lled with electrolyte [4]. Series resistance was typically between 10 and 15 MV. In voltage-clamp recordings, neurones were clamped at 260 mV and current-voltage relationships were examined using a voltage ramp protocol between 2140 and 260 mV (20 mV/s) in order to evaluate the effects of drug application. All data in the text and ®gures are presented as mean values ^ SEM unless otherwise stated. During the course of this study recordings were obtained from 58 visually identi®ed interneurones within the stratum oriens layer of the hippocampus. These neurones were typically present in low density and were found to exhibit a heterogeneous morphology. Following formation of the whole-cell con®guration, these neurones displayed electrophysiological characteristics similar to those previously attributed to GABAergic interneurones in this area [14]. For example, in current clamp recordings, these neurones had a resting membrane potential of 255:3 ^ 3:4 mV (n 58) and often ®red spontaneous action potentials at a rate of 2±10 Hz which were of short duration (1:1 ^ 0:1 ms
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 99) 0 04 49- 8
K. Lee et al. / Neuroscience Letters 270 (1999) 56±58
Fig. 1. (a) Continuous whole-cell current clamp recordings demonstrating the effect of 10 mM 5HT on an interneurone in the statum oriens layer of the hippocampus. The effect of 5HT is maintained in the presence of TTX and antagonists of ionotropic glutamate and GABAA receptors. (b) The effects of 10 mM 5HT are mimicked by the 5HT2 agonist DOI but not the 5HT3 agonist mCPBG.
measured at half-amplitude) and which were followed by a rapid after-hyperpolarization (16:1 ^ 1:3 mV in amplitude, 151:4 ^ 11:3 ms in duration). Immediately after membrane breakthrough these neurones had an input resistance of 466:5 ^ 16:2 MV (n 58) and exhibited a characteristic reduction in apparent input resistance in response to hyper-
57
polarizing current injection, previously been attributed to the non-selective cation channel current IH [6]. In current clamp recordings, bath application of 5HT (10 mM) had variable effects on these interneurones. Thus, 63% of neurones tested under these conditions were rapidly depolarized by bath application of this ligand, 26% were hyperpolarized and 11% were apparently unaffected. These effects were not correlated with any obvious morphological differences between interneurones. Since the predominant effect of this ligand was a membrane depolarization, this response was characterized in further detail. The depolarization was rapid in onset and was unaffected by the subsequent addition of 1 mM TTX together with the glutamate receptor antagonists NBQX (10 mM) and D-APV (50 mM) and the GABAA receptor antagonist bicuculline (10 mM; control: 14:2 ^ 3:4 mV depolarization; in the presence of inhibitors: 13:3 ^ 4:2 mV depolarization (n 4); Fig. 1a). Following washout of 5HT, the depolarization was found to slowly reverse over several minutes as is consistent with a G-protein mediated effect. When neurones were voltage clamped at 260 mV, 5HT was found to induce an inward current in a concentration dependent manner. Thus 100 nM 5HT had no effect on the magnitude of the holding current (n 3), 1 mM 5HT induced an inward current of 15:5 ^ 3:5 pA (n 4) whilst 10 and 20 mM 5HT induced a noisy inward current 62:2 ^ 9:5 pA (n 15; Fig. 2a) in magnitude. Using voltage ramps from 2140 to 260 mV it was possible to extrapolate a reversal potential for the 5HT-induced current of 22:1 ^ 4:1 mV (n 5; Fig. 2b). The magnitude of this current was unaffected by the introduction of a Ca-free ACSF (control
Fig. 2. (a) 5HT (10 mM) induces a ritanserin-sensitive inward current at 260 mV. (b) Expanded traces of the current responses to the voltage ramps shown in (a). The numbers beside these currents correspond to those shown in (a). (c) The inward current elicited by 5HT is reversibly inhibited by partial replacement of extracellular Na 1 ions with Tris.
58
K. Lee et al. / Neuroscience Letters 270 (1999) 56±58
71 ^ 4:1 pA; Ca-free ACSF: 66:9 ^ 3:9 pA (n 3)). To determine whether Na 1 ions were involved in the generation of this current, the external NaCl was completely replaced with Tris±HCl. Under these conditions, the amplitude of the 5HT-induced current was reduced to 36:2 ^ 6:3% its control value (n 4; Fig. 2c). The effects of 5HT were mimicked by the 5HT2 selective agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 10 mM) which induced an inward current of 55 ^ 4:5 pA in cells voltage clamped at 260 mV and a depolarization under current clamp conditions (n 3 for each; Fig. 1b). In contrast, the 5HT3 selective agonists 2-methyl-5hydroxytryptamine and m-Chlorophenylbiguanide were both without effect under current clamp conditions and in cells voltage clamped at 260 mV (n 3 for each; Fig. 1b). These ®ndings suggest that a 5HT2 receptor might be responsible for the inward current induced by 5HT in these neurones. This assumption was supported by the ®nding that the 5HT-induced inward current was abolished by pre-incubation with the 5HT2 receptor antagonist ritanserin (1 mM; n 5; Fig. 2a) [3] but was unaffected by preincubation with the 5HT3 antagonist LY278,584 (10 mM; not shown). These ®ndings demonstrate that 5HT depolarizes interneurones within the stratum oriens layer of the hippocampus via the activation of an inward current at 260 mV. This current was abolished by bath application of the 5HT2 receptor antagonist ritanserin and was mimicked by the 5HT2 receptor agonist DOI to suggest that these neurones express functional 5HT2 receptors [3]. Interneurones within the stratum oriens layer of the hippocampus have previously been shown to make inhibitory synaptic connections with CA1 pyramidal neurones and consequently, these ®ndings help reconcile recent electrophysiological ®ndings demonstrating that 5HT2 receptor stimulation within the hippocampus potentiates GABAergic IPSCs in CA1 pyramidal neurones [12]. Although in the present study we have not attempted to determine the subtype of 5HT2 receptor responsible for these effects, both 5HT2A and 5HT2C receptors have previously been shown to be expressed within the stratum oriens layer of the hippocampus [9]. In conclusion, in the present study we have demonstrated that presumed GABAergic interneurones within the stratum oriens layer of the hippocampus express functional 5HT2 receptors which when stimulated lead to neuronal excita-
tion. These ®ndings provide further information to aid our understanding of serotonergic control of hippocampal function.
[1] Acsady, L., Halasy, K. and Freund, T.F., Calretinin is present in non-pyramidal cells of the rat hippocampus- III: their inputs from the median raphe and medial septal nuclei. Neuroscience, 52 (1993) 829±841. [2] Andrade, R. and Nicoll, R.A., Pharmacologically distinct actions of serotonin on single pyramidal neurones of the rat hippocampus recorded in vitro. J. Physiol., 394 (1987) 99±124. [3] Baxter, G., Kennett, G., Blaney, F. and Blackburn, T., 5-HT2 receptor subtypes: a family re-united? Trends Pharmacol. Sci., 16 (1995) 105±110. [4] Bell, M.I., Richardson, P.J. and Lee, K., Characterization of the mechanism of action of tachykinins in rat striatal cholinergic interneurons. Neuroscience, 87 (1998) 649±658. [5] Freund, T.F., Gulyas, A.I., Acsady, L., Gores, T. and Toth, K., Serotonergic control of the hippocampus via local inhibitory interneurons. Proc. Natl. Acad. Sci. USA, 87 (1990) 8501±8505. [6] Maccaferri, G. and McBain, C.J., The hyperpolarization-activated current (IH) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones. J. Physiol., 497 (1996) 119±130. [7] McMahon, L.L. and Kauer, J.A., Hippocampal interneurons are excited via serotonin-gated ion channels. J. Neurophysiol., 78 (1997) 2493±2502. [8] Moore, R.Y. and Halaris, A.E., Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat. J. Comp. Neurol., 164 (1975) 171±184. [9] Pompeiano, M., Palacios, J.M. and Mengod, G., Distribution of the serotonin 5HT2 receptor family mRNAs: comparison between 5HT2a and 5HT2c receptors. Mol. Brain Res., 23 (1994) 163±174. [10] Ropert, N. and Guy, N., Serotonin facilitates GABAergic transmission in the CA1 region of the rat hippocampus in vitro. J. Physiol., 441 (1991) 121±136. [11] Segal, M., The action of serotonin in the rat hippocampal slice preparation. J. Physiol., 303 (1980) 423±439. [12] Shen, R.Y. and Andrade, R., 5-hydroxytryptamine2 receptor facilitates GABAergic neurotransmission in rat hippocampus. J. Pharm. Exp. Ther., 285 (1998) 805±812. [13] Stuart, G.J., Dodt, H.U. and Sakmann, B., Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. P¯ugers Arch., 423 (1993) 511±518. [14] Zhang, L. and McBain, C.J., Potassium conductances underlying repolarization and after-hyperpolarization in rat CA1 hippocampal interneurones. J. Physiol., 488 (1995) 661±672.
Serotonin depolarizes hippocampal interneurones in the rat stratum oriens by interaction with 5HT2 receptors K. Lee*, A.K. Dixon, R.D. Pinnock Parke-Davis Neuroscience Research Centre, Cambridge University Forvie Site, Robinson Way, Cambridge, CB2 2QB, UK Received 20 February 1999; received in revised form 14 May 1999; accepted 21 May 1999
Abstract Patch-clamp recording techniques were used to examine the effect of serotonin (5HT) upon interneurones contained in the stratum oriens layer of hippocampal slices. Bath application of 1±20 mM 5HT depolarized neurones by the induction of an inward current at 260 mV. This inward current was Na 1-dependent in nature, was mimicked by the 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and was inhibited by pre-incubation with the 5HT2 receptor antagonist ritanserin. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Whole-cell patch clamp; Serotonin; Hippocampus
The mammalian hippocampus receives a dense serotonergic innervation from the raphe nuclei and incoming ®bres make synaptic contacts on both pyramidal neurones and inhibitory GABAergic interneurones [1,8]. Although electrophysiological studies have revealed a variety of serotonin (5HT)-mediated responses in pyramidal neurones [2,11] much less is known about the effects of this neurotransmitter on inhibitory interneurones. Previous studies have shown that 5HT causes a large increase in spontaneous GABAergic synaptic potentials in hippocampal CA1 pyramidal neurones [5,10]. This effect is thought to arise at least in part through 5HT3 mediated depolarization of interneurones in the stratum lacunosum [7]. However, Shen and Andrade [12] have recently provided evidence to suggest that 5HT2 receptor stimulation also enhances GABAergic transmission in the hippocampus. In the present study we demonstrate that these effects may arise from interneurones situated within the stratum oriens layer. Brains were removed from 14±28 day-old male Wistar rats and 300 mm coronal slices containing the hippocampus were prepared in physiological saline using a vibratome. In all experiments neurones were visualized using infrared differential interference contrast videomicroscopy [13]. The physiological saline contained (mM) 125.0 NaCl, 25.0 NaHCO3, 10.0 glucose, 2.5 KCl, 1.25 NaH2PO4, 2.0 * Corresponding author. Tel.: 144-1223-210-929; fax: 1441223-249-106. E-mail address: [email protected] (K. Lee)
CaCl2, 1.0 MgCl2 and was bubbled with a 95%, 5% O2/CO2 gas mixture. This solution was also used as the extracellular solution in all experiments. The intracellular (pipette) solution comprised (mM) 120.0 Kgluconate, 10.0 NaCl, 2.0 MgCl2, 1.0 K2EGTA, 10.0 HEPES, 4.0 Na2ATP, 0.3 Na2GTP, pH adjusted to 7.2 with KOH. All experiments were conducted at 30±358C. Electrophysiological recordings were performed as previously described using borosilicate glass recording electrodes with resistances of 3±6 MV when ®lled with electrolyte [4]. Series resistance was typically between 10 and 15 MV. In voltage-clamp recordings, neurones were clamped at 260 mV and current-voltage relationships were examined using a voltage ramp protocol between 2140 and 260 mV (20 mV/s) in order to evaluate the effects of drug application. All data in the text and ®gures are presented as mean values ^ SEM unless otherwise stated. During the course of this study recordings were obtained from 58 visually identi®ed interneurones within the stratum oriens layer of the hippocampus. These neurones were typically present in low density and were found to exhibit a heterogeneous morphology. Following formation of the whole-cell con®guration, these neurones displayed electrophysiological characteristics similar to those previously attributed to GABAergic interneurones in this area [14]. For example, in current clamp recordings, these neurones had a resting membrane potential of 255:3 ^ 3:4 mV (n 58) and often ®red spontaneous action potentials at a rate of 2±10 Hz which were of short duration (1:1 ^ 0:1 ms
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 99) 0 04 49- 8
K. Lee et al. / Neuroscience Letters 270 (1999) 56±58
Fig. 1. (a) Continuous whole-cell current clamp recordings demonstrating the effect of 10 mM 5HT on an interneurone in the statum oriens layer of the hippocampus. The effect of 5HT is maintained in the presence of TTX and antagonists of ionotropic glutamate and GABAA receptors. (b) The effects of 10 mM 5HT are mimicked by the 5HT2 agonist DOI but not the 5HT3 agonist mCPBG.
measured at half-amplitude) and which were followed by a rapid after-hyperpolarization (16:1 ^ 1:3 mV in amplitude, 151:4 ^ 11:3 ms in duration). Immediately after membrane breakthrough these neurones had an input resistance of 466:5 ^ 16:2 MV (n 58) and exhibited a characteristic reduction in apparent input resistance in response to hyper-
57
polarizing current injection, previously been attributed to the non-selective cation channel current IH [6]. In current clamp recordings, bath application of 5HT (10 mM) had variable effects on these interneurones. Thus, 63% of neurones tested under these conditions were rapidly depolarized by bath application of this ligand, 26% were hyperpolarized and 11% were apparently unaffected. These effects were not correlated with any obvious morphological differences between interneurones. Since the predominant effect of this ligand was a membrane depolarization, this response was characterized in further detail. The depolarization was rapid in onset and was unaffected by the subsequent addition of 1 mM TTX together with the glutamate receptor antagonists NBQX (10 mM) and D-APV (50 mM) and the GABAA receptor antagonist bicuculline (10 mM; control: 14:2 ^ 3:4 mV depolarization; in the presence of inhibitors: 13:3 ^ 4:2 mV depolarization (n 4); Fig. 1a). Following washout of 5HT, the depolarization was found to slowly reverse over several minutes as is consistent with a G-protein mediated effect. When neurones were voltage clamped at 260 mV, 5HT was found to induce an inward current in a concentration dependent manner. Thus 100 nM 5HT had no effect on the magnitude of the holding current (n 3), 1 mM 5HT induced an inward current of 15:5 ^ 3:5 pA (n 4) whilst 10 and 20 mM 5HT induced a noisy inward current 62:2 ^ 9:5 pA (n 15; Fig. 2a) in magnitude. Using voltage ramps from 2140 to 260 mV it was possible to extrapolate a reversal potential for the 5HT-induced current of 22:1 ^ 4:1 mV (n 5; Fig. 2b). The magnitude of this current was unaffected by the introduction of a Ca-free ACSF (control
Fig. 2. (a) 5HT (10 mM) induces a ritanserin-sensitive inward current at 260 mV. (b) Expanded traces of the current responses to the voltage ramps shown in (a). The numbers beside these currents correspond to those shown in (a). (c) The inward current elicited by 5HT is reversibly inhibited by partial replacement of extracellular Na 1 ions with Tris.
58
K. Lee et al. / Neuroscience Letters 270 (1999) 56±58
71 ^ 4:1 pA; Ca-free ACSF: 66:9 ^ 3:9 pA (n 3)). To determine whether Na 1 ions were involved in the generation of this current, the external NaCl was completely replaced with Tris±HCl. Under these conditions, the amplitude of the 5HT-induced current was reduced to 36:2 ^ 6:3% its control value (n 4; Fig. 2c). The effects of 5HT were mimicked by the 5HT2 selective agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 10 mM) which induced an inward current of 55 ^ 4:5 pA in cells voltage clamped at 260 mV and a depolarization under current clamp conditions (n 3 for each; Fig. 1b). In contrast, the 5HT3 selective agonists 2-methyl-5hydroxytryptamine and m-Chlorophenylbiguanide were both without effect under current clamp conditions and in cells voltage clamped at 260 mV (n 3 for each; Fig. 1b). These ®ndings suggest that a 5HT2 receptor might be responsible for the inward current induced by 5HT in these neurones. This assumption was supported by the ®nding that the 5HT-induced inward current was abolished by pre-incubation with the 5HT2 receptor antagonist ritanserin (1 mM; n 5; Fig. 2a) [3] but was unaffected by preincubation with the 5HT3 antagonist LY278,584 (10 mM; not shown). These ®ndings demonstrate that 5HT depolarizes interneurones within the stratum oriens layer of the hippocampus via the activation of an inward current at 260 mV. This current was abolished by bath application of the 5HT2 receptor antagonist ritanserin and was mimicked by the 5HT2 receptor agonist DOI to suggest that these neurones express functional 5HT2 receptors [3]. Interneurones within the stratum oriens layer of the hippocampus have previously been shown to make inhibitory synaptic connections with CA1 pyramidal neurones and consequently, these ®ndings help reconcile recent electrophysiological ®ndings demonstrating that 5HT2 receptor stimulation within the hippocampus potentiates GABAergic IPSCs in CA1 pyramidal neurones [12]. Although in the present study we have not attempted to determine the subtype of 5HT2 receptor responsible for these effects, both 5HT2A and 5HT2C receptors have previously been shown to be expressed within the stratum oriens layer of the hippocampus [9]. In conclusion, in the present study we have demonstrated that presumed GABAergic interneurones within the stratum oriens layer of the hippocampus express functional 5HT2 receptors which when stimulated lead to neuronal excita-
tion. These ®ndings provide further information to aid our understanding of serotonergic control of hippocampal function.
[1] Acsady, L., Halasy, K. and Freund, T.F., Calretinin is present in non-pyramidal cells of the rat hippocampus- III: their inputs from the median raphe and medial septal nuclei. Neuroscience, 52 (1993) 829±841. [2] Andrade, R. and Nicoll, R.A., Pharmacologically distinct actions of serotonin on single pyramidal neurones of the rat hippocampus recorded in vitro. J. Physiol., 394 (1987) 99±124. [3] Baxter, G., Kennett, G., Blaney, F. and Blackburn, T., 5-HT2 receptor subtypes: a family re-united? Trends Pharmacol. Sci., 16 (1995) 105±110. [4] Bell, M.I., Richardson, P.J. and Lee, K., Characterization of the mechanism of action of tachykinins in rat striatal cholinergic interneurons. Neuroscience, 87 (1998) 649±658. [5] Freund, T.F., Gulyas, A.I., Acsady, L., Gores, T. and Toth, K., Serotonergic control of the hippocampus via local inhibitory interneurons. Proc. Natl. Acad. Sci. USA, 87 (1990) 8501±8505. [6] Maccaferri, G. and McBain, C.J., The hyperpolarization-activated current (IH) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones. J. Physiol., 497 (1996) 119±130. [7] McMahon, L.L. and Kauer, J.A., Hippocampal interneurons are excited via serotonin-gated ion channels. J. Neurophysiol., 78 (1997) 2493±2502. [8] Moore, R.Y. and Halaris, A.E., Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat. J. Comp. Neurol., 164 (1975) 171±184. [9] Pompeiano, M., Palacios, J.M. and Mengod, G., Distribution of the serotonin 5HT2 receptor family mRNAs: comparison between 5HT2a and 5HT2c receptors. Mol. Brain Res., 23 (1994) 163±174. [10] Ropert, N. and Guy, N., Serotonin facilitates GABAergic transmission in the CA1 region of the rat hippocampus in vitro. J. Physiol., 441 (1991) 121±136. [11] Segal, M., The action of serotonin in the rat hippocampal slice preparation. J. Physiol., 303 (1980) 423±439. [12] Shen, R.Y. and Andrade, R., 5-hydroxytryptamine2 receptor facilitates GABAergic neurotransmission in rat hippocampus. J. Pharm. Exp. Ther., 285 (1998) 805±812. [13] Stuart, G.J., Dodt, H.U. and Sakmann, B., Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. P¯ugers Arch., 423 (1993) 511±518. [14] Zhang, L. and McBain, C.J., Potassium conductances underlying repolarization and after-hyperpolarization in rat CA1 hippocampal interneurones. J. Physiol., 488 (1995) 661±672.