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J Neurophysiol 112: 30 –38, 2014. First published April 16, 2014; doi:10.1152/jn.00582.2013. Endogenous release of 5-HT modulates the plateau phase of NMDA-induced membrane potential oscillations in lamprey spinal neurons Di Wang,1,2 Sten Grillner,2 and Peter Wallén2 1 Department of Physiology, Liaoning Medical University, Jinzhou, People’s Republic of China; and 2Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Submitted 15 August 2013; accepted in final form 15 April 2014 plateau properties; pacemaker-like oscillations; spinal serotonin system; calcium-dependent potassium channels; locomotor network IN ALL VERTEBRATES INVESTIGATED, the networks underlying locomotion are located in the spinal cord, while they are activated by glutamatergic reticulospinal neurons in the brain stem, acting through ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartic acid (NMDA) receptors (Grillner 2006; Ohta and Grillner 1989). Descending pathways and intraspinal neurons using 5-HT as a transmitter also play an important role in many vertebrates (Jordan et al. 2008; Schotland et al. 1995). 5-HT acts through voltagedependent calcium channels, which in turn, control the postspike afterhyperpolarization via calcium-dependent potassium (KCa) channels [lamprey: Hill et al. (2003) and Wallén et al. Address for reprint requests and other correspondence: P. Wallén, Nobel Institute for Neurophysiology, Dept. of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden (e-mail: [email protected]). 30 (1989); rat: Bayliss et al. (1995) and Blomeley and Bracci (2005); turtle: Hounsgaard and Kiehn (1989); frog: Scroggs and Anderson (1989) and Sun and Dale (1998)] and also acts through presynaptic inhibition [lamprey: Buchanan and Grillner (1991) and Schwartz et al. (2005); mouse: Garcia-Ramirez et al. (2014); frog: Sillar and Simmers (1994b)]. In the lamprey, administration of 5-HT agonists slows down the locomotor activity and makes the bursts more intense (Harris-Warrick and Cohen 1985). Conversely, administration of 5-HT antagonists during locomotor activity leads to a pronounced increase of the locomotor frequency (Zhang and Grillner 2000). In the lamprey, the locomotor networks can be activated by superfusion of NMDA to the isolated spinal cord preparation (Grillner et al. 1981). The motor activity can be recorded from the ventral roots or from motoneurons and interneurons intracellularly. The key elements of the segmental and intersegmental networks responsible for generating the locomotor activity have been identified, and they consist of ipsilateral excitatory interneurons and commissural inhibitory interneurons (Buchanan and Grillner 1987; Grillner 2003; Kozlov et al. 2009). In the isolated spinal cord, after cellular interactions have been blocked through administration of TTX, the activation of NMDA receptors generates pacemaker-like oscillations in many neurons, which rely on the voltage-dependent properties of NMDA receptors in interaction with voltage-dependent potassium and KCa channels (Wallén and Grillner 1987). NMDA receptor-mediated, TTX-resistant membrane potential oscillations can contribute to the generation of locomotor activity and have also been demonstrated in other species, including rodents (Hochman et al. 1994; MacLean et al. 1997; Masino et al. 2012; Schmidt et al. 1998) and amphibians (Li et al. 2010; Reith and Sillar 1998; Scrymgeour-Wedderburn et al. 1997). In the lamprey, these oscillations vary in a characteristic way between different spinal cord preparations. In some preparations, there is a pronounced, depolarized plateau phase, which can be prolonged further by a blockade of KCa channels via administration of 5-HT or specific antagonists, such as apamin (El Manira et al. 1994). In other preparations, there are instead more short-lasting depolarizations without any plateau phase [cf. Wang et al. (2013)], lasting approximately 200 –300 ms. In the present study, we characterize these two types of oscillations and show that they depend on the level of endogenous release of 5-HT in the spinal cord preparations. The plateaulike oscillations are converted to the second type of oscillations lacking a plateau phase when 5-HT antagonists are administered, and conversely, plateau-like oscillations can be induced or prolonged by 5-HT agonists. These properties most likely 0022-3077/14 Copyright © 2014 the American Physiological Society www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 18, 2017 Wang D, Grillner S, Wallén P. Endogenous release of 5-HT modulates the plateau phase of NMDA-induced membrane potential oscillations in lamprey spinal neurons. J Neurophysiol 112: 30 –38, 2014. First published April 16, 2014; doi:10.1152/jn.00582.2013.— The lamprey central nervous system has been used extensively as a model system for investigating the networks underlying vertebrate motor behavior. The locomotor networks can be activated by application of glutamate agonists, such as N-methyl-D-aspartic acid (NMDA), to the isolated spinal cord preparation. Many spinal neurons are capable of generating pacemaker-like membrane potential oscillations upon activation of NMDA receptors. These oscillations rely on the voltage-dependent properties of NMDA receptors in interaction with voltage-dependent potassium and calcium-dependent potassium (KCa) channels, as well as low voltage-activated calcium channels. Upon membrane depolarization, influx of calcium will activate KCa channels, which in turn, will contribute to repolarization and termination of the depolarized phase. The appearance of the NMDAinduced oscillations varies markedly between spinal cord preparations; they may either have a pronounced, depolarized plateau phase or be characterized by a short-lasting depolarization lasting approximately 200 –300 ms without a plateau. Both types of oscillations increase in frequency with increased concentrations of NMDA. Here, we characterize these two types of membrane potential oscillations and show that they depend on the level of endogenous release of 5-HT in the spinal cord preparations. In the lamprey, 5-HT acts to block voltage-dependent calcium channels and will thereby modulate the activity of KCa channels. When 5-HT antagonists were administered, the plateau-like oscillations were converted to the second type of oscillations lacking a plateau phase. Conversely, plateau-like oscillations can be induced or prolonged by 5-HT agonists. These properties are most likely of significance for the modulatory action of 5-HT on the spinal networks for locomotion. ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY play a role in the generation of locomotor activity by the spinal networks. MATERIALS AND METHODS ventral roots were drawn into the tips of glass suction electrodes for motoneuron identification. The chamber was perfused continuously with oxygenated lamprey physiological solution containing (in mM): 138 NaCl, 2.1 KCl, 1.8 CaCl2, 1.2 MgCl2, 4 glucose, 2 HEPES, and 0.5 L-glutamine, bubbled with O2 and adjusted to pH 7.4 with NaOH. The experimental chamber was cooled to maintain the preparation at 6 –10°C. Drugs used were NMDA (Tocris, Bristol, UK); TTX (final concentration 1.5 M; Sigma); 5-HT (1 M; Tocris), spiperone hydrochloride, a 5-HT antagonist (10 M; Tocris); NAD 299 hydrochloride, a specific 5-HT1A antagonist [(3R)-3-(dicyclobutylamino)-8-fluoro-3,4-dihydro-2H1-benzopyran-5-carboxamide hydrochloride, 10 –20 M; Tocris]; and nimodipine, an L-type calcium channel antagonist [1,4-dihydro-2,6dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 2-methyloxyethyl 1-methylethyl ester, 10 M; Tocris]. All drugs were applied to the perfusion solution. NMDA was applied in incremental concentration steps of 50 M, from 50 to 200 M or in one case, to 250 M. Electrophysiology. To induce NMDA receptor-dependent, TTXresistant oscillations, NMDA and TTX were added in the physiological solution (Wallén and Grillner 1987). For intracellular recordings, sharp microelectrodes were filled with 3 M KAc and 0.1 M KCl B -48 2 1 100µM NMDA oscillation frequency (Hz) A 150µM NMDA mV -70 3 200µM NMDA C 10s 4 250µM NMDA 8 ½ 100µM 150µM 200µM Width 0.0 35 30 amplitude (mV) cycle duration (s) 0.2 100 150 200 NMDA concentration (µM) 2 Amplitude 6 4 2 25 20 15 10 5 0 0 cell 2 cell 3 cell 4 cell 5 5 4 3 2 1 0 4 1 half-width/cycle duration cell 1 half-width (s) 0.4 Cycle duration 1 10 3 0.6 0.5 cell 1 cell 2 cell 3 cell 4 cell 5 cell 1 cell 2 cell 3 cell 4 cell 5 0 cell 1 cell 2 cell 3 cell 4 cell 5 Fig. 1. Membrane potential oscillations depend on the N-methyl-D-aspartic acid (NMDA) concentration. A: NMDA-induced, TTX-resistant membrane potential oscillations at different concentrations of NMDA in a lamprey spinal neuron, recorded in discontinuous current clamp mode. The average trough membrane potential (⫺70 mV) was kept constant by direct current (DC)-current injection. The oscillation frequency was increased markedly when concentration was raised from 100 to 150 M (A1 and A2). At 200 and 250 M (A3 and A4), no further change of oscillation frequency was apparent. Time and voltage calibrations in A1 apply to all records. B: relation between NMDA concentration and oscillation frequency. Combined results from 9 neurons tested. The overall frequency range was 0.08 – 0.54 Hz. The higher doses resulted in higher frequency, with the more marked increase commonly occurring from 100 to 150 M. In several cells, the dose-frequency relation tended to saturate at the highest dose (200 M). Each point represents the average frequency from 10 cycles at each concentration in an individual neuron. C: the influence of changing the NMDA concentration on the structure of the oscillatory cycle. Inset: the different parameters measured: cycle duration, time from 1 depolarization peak to the next; amplitude, voltage deflection from trough to peak; half-width, duration of depolarized plateau at half-maximal amplitude (½). These parameters were analyzed in 5 cells with plateau-like oscillations: cycle duration (C1), amplitude (C2), half-width (C3), and half-width normalized to cycle duration (C4). Average values from 20 cycles recorded at each concentration. Student’s t-test, means ⫾ SD, comparisons of nearby groups. J Neurophysiol • doi:10.1152/jn.00582.2013 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 18, 2017 Ethical approval. All experimental procedures were approved by Stockholm Norra Försöksdjursetiska Nämnd, according to the Swedish regulations for the care and use of laboratory animals. Before removal of the tissue, the animals were deeply anesthetized (MS-222, tricaine methanesulfonate, 100 mg/l, dissolved in water; Sigma, St. Louis, MO) and killed by decapitation. In vitro lamprey spinal cord preparation. Experiments were performed using isolated spinal cord preparations of adult river lampreys (Lampetra fluviatilis; n ⫽ 31) or in a few cases, American silver lampreys (Ichthyomyzon unicuspis; n ⫽ 2). Results obtained from the two species were indistinguishable. In previous studies, no differences in cellular properties of spinal neurons in the two species have been observed. A spinal cord piece comprising 10 –15 segments was dissected from a region caudal to the gills. The spinal cord was isolated from the notochord and placed with the ventral side up into a Sylgard-lined (Dow Corning, Midland, MI) recording chamber. The meninges were stripped from the ventral surface, and in most cases, 31 32 ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY (resistance 30 –50 M⍀). Cells were recorded in current clamp mode using an AxoClamp 2A amplifier (Axon Instruments, Weatherford, TX). The general membrane potential level was adjusted by direct current injection when needed. In cases when recordings were started before TTX application, motoneurons could be identified by recording their axonal spike in the ventral root. Student’s t-test was used for statistical comparisons of oscillation cycle parameters (cf. Fig. 1C1, inset). Parameter values are given as means ⫾ SD, with levels of significance: *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001. RESULTS A 1 100µM NMDA 3 200µM NMDA 2 150µM NMDA -30 mV -60 10s B 1 2 25 100µM 150µM 200µM 20 35 30 amplitude(mV) cycle duration(s) 25 15 10 20 15 10 5 5 0 0 cell 6 3 cell 7 cell 8 cell 9 cell 10 1 cell 6 cell 7 cell 8 cell 9 cell 10 cell 6 cell 7 cell 8 cell 9 cell 10 4 0.5 half-width/cycle duration 0.4 half-width(s) Fig. 2. Lamprey spinal neurons may generate spike-like oscillations that are also influenced by the NMDA level. A: a lamprey spinal neuron generating spike-like NMDA oscillations. The oscillation frequency was increased markedly when NMDA concentration was raised from 100 (A1) to 150 M (A2). At 200 M NMDA (A3), oscillation amplitude increased further, whereas the frequency did not. Time and voltage calibrations in A1 apply to all records. B1–B4: the influence of changing the NMDA concentration on the structure of the oscillatory cycle in 5 neurons with spike-like oscillations. Parameters analyzed were as in Fig. 1C. The effects of varying the NMDA concentration on the cycle structure were similar in the 2 types of oscillations. Average values from 20 cycles recorded at each concentration. Student’s t-test, means ⫾ SD, comparisons of nearby groups. 0.5 0.3 0.2 0.1 0 0 cell 6 cell 7 cell 8 cell 9 cell 10 J Neurophysiol • doi:10.1152/jn.00582.2013 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 18, 2017 Upon application of NMDA and in the presence of TTX, many neurons in the lamprey spinal cord will generate endogenous, pacemaker-like membrane potential oscillations (Wallén and Grillner 1985, 1987). These oscillations are strongly voltage dependent, and previous reports have also suggested a positive correlation between the frequency of the oscillations and the NMDA concentration (Huss et al. 2008; Sigvardt et al. 1985; Wallén and Grillner 1987). The NMDA-induced, TTX-resistant membrane potential oscillations vary in shape between different spinal cord preparations; the oscillations may be “plateau like” with a pronounced, depolarized plateau phase (cf. Fig. 1A) but may in other preparations appear “spike like” with a short-lasting depolarization and no evident plateau phase (cf. Fig. 2A). Below, we will characterize these two forms of oscillation by a quantitative analysis of the effects of varying the NMDA concentra- tion. In addition, we describe the modulatory influence of endogenous 5-HT release on the oscillations. Dependence of the membrane potential oscillations on NMDA concentration. In most cells of the lamprey spinal cord, the threshold concentration of NMDA to induce membrane potential oscillations is approximately 50 –100 M (Wallén and Grillner 1987). Figure 1A illustrates one neuron where 100 M NMDA induced rhythmic plateau oscillations at a rate of ⬃0.2 Hz (Fig. 1A1). Increasing the NMDA concentration to 150 M (Fig. 1A2) caused an increase in frequency to ⬃0.25 Hz, whereas a higher concentration did not result in any obvious, further increase in the rate of oscillations (Fig. 1, A3 and A4). In general, there was a tendency to reach “saturation” ⬃150 M, with no further increase in the rate of oscillations with higher levels of NMDA (Fig. 1B). During NMDA-induced fictive locomotor activity in the spinal network, a corresponding relation is seen (Brodin et al. 1985). The influence of varying the NMDA concentration on different parameters characterizing the oscillatory behavior was analyzed further in 10 spinal cord neurons (of which three were identified as motoneurons), with five cells showing plateau-like oscillations (Fig. 1C) and the other five cells showing spikelike oscillations (Fig. 2B). For plateau-like oscillations, the cycle duration (see inset in Fig. 1C1 depicting measured parameters) was reduced significantly, with higher NMDA concentration in three of the five cells (Fig. 1C1), whereas one cell (cell 4) showed a change in the opposite direction. The ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY -40 B 2 150µM NMDA 1 100µM NMDA 0.50 oscillation frequency (Hz) A sponding to a range between 2% and 10% of the cycle (Fig. 2B4). Higher NMDA concentrations caused a significant increase of the normalized half-width in all five tested cells with spike-like oscillations (Fig. 2B4). Even so, the half-width values were typically ⬍10% of the cycle even with higher levels of NMDA. The NMDA-induced oscillations are strongly dependent on the membrane potential level (Wallén and Grillner 1985, 1987). We therefore also analyzed the effects of varying the NMDA concentration at two different holding potential levels (n ⫽ 5; Fig. 3). Figure 3A illustrates spike-like oscillations recorded at a holding potential of ⫺70 mV (Fig. 3, A1 and A2) and at ⫺75 mV (Fig. 3, A3 and A4) and with two different levels of NMDA. Increasing the concentration of NMDA increased the oscillation frequency at both holding potential levels, but the effect was more prominent at the ⫺70-mV level, thus giving a steeper relation between NMDA concentration and oscillation frequency (Fig. 3B). Corresponding results were also found in four neurons displaying plateau-like oscillations (not illustrated). The effects on the structure of the oscillation cycle of the cell in Fig. 3, A and B, are illustrated in Fig. 3C. Corresponding to the graph in Fig. 3B, the cycle duration (Fig. 3C1) was significantly longer at the more hyperpolarized level with the higher NMDA concentration (150 M), whereas with 100 M NMDA, there was no difference. The oscillation amplitude (Fig. 3C2) was significantly larger at ⫺75 mV and with both NMDA concentrations. The half-width (Fig. 3C3) did not mV -80 Holding pot.-70mV 10s Holding pot.-70mV 4 150µM NMDA 3 100µM NMDA -70mV -75mV 0.25 0.00 100 150 NMDA concentration (µM) Holding pot.-75mV 100µM 150µM 1 2 40 35 +++ 6 4 amplitude (mV) cycle duration (s) 8 +++ ++ 0.3 30 25 20 15 10 2 0.2 0.1 5 0 0 -70mV -75mV 0 -70mV -75mV +++ 4 3 half-width (s) C half-width/cycle duration Holding pot.-75mV 0.10 0.06 0.02 0 -70mV -75mV -70mV -75mV Fig. 3. NMDA oscillations depend on both NMDA concentration and membrane potential. A: at both holding potentials (Holding pot.), ⫺70 mV (A1 and A2) and ⫺75 mV (A3 and A4), oscillation frequency increased with NMDA concentration. At the higher NMDA level (150 M), depolarization of the holding potential also caused a marked frequency increase (A2 and A4). Time and voltage calibrations in A1 apply to all records. B: relation between NMDA concentration and oscillation frequency in the same neuron as in A. Each point represents the frequency from 10 cycles. At the more depolarized holding potential level, the relation is steeper. C: cycle structure analysis of oscillations in the same neuron as in A (cf. Fig. 1C1, inset). Average values from 20 cycles of oscillation. Cycle duration (C1) was increased significantly at the more hyperpolarized level and with the higher NMDA concentration, whereas the amplitude (C2) increased significantly upon hyperpolarization with both high and low NMDA concentration. Half-width (C3 and C4) was decreased significantly at the more hyperpolarized level with 150 M NMDA but only when expressed as normalized values (C4). Student’s t-test, means ⫾ SD; comparison of 100 M group vs. 150 M group: *P ⬍ 0.05, ***P ⬍ 0.001; comparison of ⫺70 mV vs. ⫺75 mV group: ⫹⫹P ⬍ 0.01, ⫹⫹⫹P ⬍ 0.001. J Neurophysiol • doi:10.1152/jn.00582.2013 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 18, 2017 trough-to-peak amplitude, ranging between 13 and 28 mV, showed a variable dependence on NMDA concentration among the cells (Fig. 1C2). The width of the depolarized plateau at half-maximal amplitude (half-width; see inset in Fig. 1C1) was typically ⬃1 s or longer and showed an increase with higher NMDA levels in four cells and a decrease in one neuron (Fig. 1C3). Normalized to the cycle duration, the half-width ranged between ⬃13% and 57% of the cycle duration (Fig. 1C4). NMDA-induced oscillations characterized by shorter depolarization peaks, without a prolonged plateau, are illustrated in Fig. 2. The spike-like oscillations also increased in frequency, with higher concentrations of NMDA (Fig. 2A). As with plateau-like oscillations, a saturation level was generally seen for spike-like oscillations in the relationship between NMDA concentration and rhythm frequency. In general, the cycle durations as well as the amplitudes were within the same range for cells with plateau- and spike-like oscillations. The effects of varying the NMDA concentration on different oscillatory parameters were also similar in cells displaying spike-like oscillations (Fig. 2B) compared with those described above for plateau-like oscillations. In all of the five cells analyzed, cycle duration became significantly shorter with higher concentrations of NMDA (Fig. 2B1). Four of the five cells showed a significant increase in oscillation amplitude with higher NMDA levels (Fig. 2B2). The brief depolarization peaks of spike-like oscillations consequently occupied a small proportion of the cycle, with the half-width varying between 0.2 and 0.4 s at the lowest NMDA concentration (Fig. 2B3), corre- 33 ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY A 1 2 Control (150µM NMDA) B 1 10µM spiperone 24min 2 2.5 -35 half-width/cycle duration 34 -70 3 10µM spiperone 40min 5s 4 10µM spiperone + 1µM 5-HT 12min half-width (s) 2 mV 1.5 1 0.5 0 C 2 Control (150µM NMDA) half-width/cycle duration 5s 4 10µM NAD + 1µM 5-HT 41min half-width (s) 10µM NAD + 1µM 5-HT 27min Control spip spip + 5-HT 0.3 mV 3 0.1 2 1.2 -65 0.2 00 spip + 5-HT D 1 10µM NAD 22min 0.3 0.8 0.4 0 Control NAD NAD + 5-HT 0.2 0.1 0 Control NAD NAD + 5-HT Fig. 4. Plateau-like oscillations are converted to the spike-like form after application of 5-HT antagonists. A and B: effects of spiperone. A1: control; plateau-like membrane potential oscillations induced by bath application of 150 M NMDA in the presence of TTX. A2 and A3: after the application of spiperone (10 M), the depolarized plateaus were gradually shortened to finally become spike like (A3). A4: the spike-like oscillations were converted back to the plateau-like form after application of 5-HT (1 M). B: effects on the half-width of the depolarized phase of the cell in A. Spiperone (spip) significantly reduced the half-width duration (B1: from 1.86 s to 0.07 s; B2: normalized values, from 0.40 to 0.02), whereas the addition of 5-HT partly counteracted the effect. Average values from 10 to 22 cycles. Student’s t-test, means ⫾ SD, comparisons of nearby groups. C and D: effects of the 5-HT1A receptor antagonist NAD 299. C1: control; plateau-like membrane potential oscillations induced by bath application of 150 M NMDA in the presence of TTX. C2: after application of NAD 299 (10 M), the plateau-like oscillations were modified to become spike like, and upon addition of 5-HT (1 M; C3 and C4), they were converted back to the plateau-like form. D: effects on the half-width of the depolarized phase of the cell in C. NAD 299 significantly reduced the half-width duration (D1: from 0.95 s to 0.48 s; D2: normalized values, from 0.22 to 0.14), whereas the addition of 5-HT counteracted the effect. Average values from 17 to 24 cycles. Student’s t-test, means ⫾ SD, comparisons of nearby groups. Traces in A and C, respectively, are from continuous recordings in discontinuous current clamp mode and with the trough membrane potential held at ⫺70 mV (A) and ⫺66 mV (C) by DC-current injection. Time and voltage calibrations in A1 apply to all traces in A, and calibrations in C1 apply to all traces in C. change with the holding potential, except when expressed as normalized half-width and with the higher NMDA concentration (Fig. 3C4). A general conclusion from these findings is thus that for both types of membrane potential oscillations, higher levels of NMDA receptor activation will result in an increased oscillation frequency until a saturation level is reached. Also, in most cases, there is an increase in oscillation amplitude and in half-width duration with higher NMDA concentrations. Serotonergic modulation of membrane potential oscillations. It is thus clear that the NMDA-induced membrane potential oscillations may vary between different spinal cord preparations. In some, the oscillations are plateau like, whereas in other preparations, there are instead spike-like oscillations, lasting approximately 200 –300 ms without any plateau phase. One may then ask whether this difference reflects different states of the preparations and if the two forms of oscillations may occur in the same cell. Differences in the functional state may be caused by influence from various modulatory systems. The duration of the depolarized plateau is prolonged by a partial blockage of KCa channels by apamin, and these channels are also depressed by 5-HT (El Manira et al. 1994; Wallén et al. 1989; Wallén and Grillner 1987). The possibility that the oscillations may be subject to an endogenous modulation by the spinal 5-HT system was first investigated by bath application of spiperone, a 5-HT antagonist (Hoyer et al. 1994; Metwally et al. 1998; Zhang and Grillner 2000). Spiperone was previously shown to increase the frequency of fictive locomotion induced by NMDA, as well as the variability of the rhythmic activity (Zhang and Grillner 2000), indicating an endogenous release of 5-HT during fictive locomotion. When spiperone was applied during plateau-like oscillations (Fig. 4, A and B), the plateau phase became progressively shorter (Fig. 4A2), and eventually, the oscillations were converted to the spike-like form, with no plateau phase (Fig. 4A3; n ⫽ 6). When 5-HT was then applied, the oscillations partly reverted back to the plateau-like form (Fig. 4A4). Spiperone thus caused a marked and significant reduction in the halfwidth of the depolarized phase (Fig. 4B), seen in all six cells tested. Due to the possibility that spiperone may, in addition, act as a dopamine receptor antagonist (Metwally et al. 1998; Seeman and Van Tol 1994), we also tested the effect of the specific 5-HT1A antagonist NAD 299 (Johansson et al. 1997) (Fig. 4, C and D; n ⫽ 6), which like spiperone, altered the plateau-like oscillations to become more spike like (Fig. 4C2), J Neurophysiol • doi:10.1152/jn.00582.2013 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 18, 2017 1 -45 Control spip 0.4 ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY C A 1 -50 Control (150µM NMDA) -55 mV 1 Control (150µM NMDA) mV -70 5s -75 5s 2 1µM 5-HT 5min 2 1µM 5-HT 9min 3 1µM 5-HT + 10µM spiperone 5min 3 1µM 5-HT + 20µM NAD 19min D 1 0.8 0.4 0 0.2 0.1 1.2 0.8 0.4 0 Control 5-HT 5-HT + spip 2 2 1.6 half-width (s) half-width/cycle duration 2 0.3 half-width/cycle duration B 1 1.2 half-width (s) plateau-like oscillations, an effect that was markedly counteracted by subsequent application of either of the two antagonists (Fig. 5, B and D). Thus 5-HT will act to promote a transition from spike-like to plateau-like oscillations, whereas blocking the influence of 5-HT will convert the oscillations back to the spike-like form. An altered level of 5-HT tone in the spinal cord may thus modulate the oscillatory activity and cause a switch from one form of oscillation to the other. 5-HT indirectly blocks KCa channels through an action on voltage-dependent calcium channels (Hill et al. 2003). Moreover, 5-HT acts on low-threshold L-type calcium channels (CaV 1.3) to modulate the postinhibitory rebound (PIR) response in lamprey spinal neurons (Wang et al. 2011). We have reported recently that during NMDA-induced membrane potential oscillations, low voltage-activated (LVA) calcium channels of the CaV 1.3 subtype will contribute (Wang et al. 2013). Control 5-HT 5-HT + spip 0 Control 5-HT 5-HT + NAD 0.4 0.3 0.2 0.1 0 Control 5-HT 5-HT + NAD Fig. 5. Spike-like oscillations are converted to the plateau-like form after application of 5-HT. A: 5-HT application, followed by addition of spiperone. A1: control; spike-like membrane potential oscillations induced by bath application of 150 M NMDA in the presence of TTX. A2: after the application of 5-HT (1 M), the oscillation peaks were prolonged to become plateau like. A3: the plateau-like oscillations were converted back to the spike-like form after addition of spiperone (10 M). B: effects on the half-width of the depolarized phase of the cell in A. 5-HT significantly increased the half-width duration (B1: from 0.21 s to 0.94 s; B2: normalized values, from 0.09 to 0.24), whereas the addition of spiperone abolished the effect. Average values from 7 to 10 cycles. Student’s t-test, means ⫾ SD, comparisons of nearby groups. C: 5-HT application, followed by addition of NAD 299. C1: control; spike-like membrane potential oscillations induced by bath application of 150 M NMDA in the presence of TTX. C2: after the application of 5-HT (1 M), oscillations were converted to become plateau like. C3: the plateau-like oscillations were converted back to the spike-like form after addition of NAD 299 (20 M). D: effects on the half-width of the depolarized phase of the cell in C. 5-HT significantly increased the half-width duration (D1: from 0.25 s to 1.36 s; D2: normalized values, from 0.07 to 0.32), whereas the addition of NAD 299 counteracted the effect. Average values from 6 to 19 cycles. Student’s t-test, means ⫾ SD, comparisons of nearby groups. Traces in A and C, respectively, are from continuous recordings in discontinuous current clamp mode and with the trough membrane potential held at ⫺70 mV (A) and ⫺74 mV (C) by DC-current injection. Time and voltage calibrations in A1 apply to all traces in A, and calibrations in C1 apply to all traces in C. J Neurophysiol • doi:10.1152/jn.00582.2013 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 18, 2017 and after subsequent application of 5-HT, the plateau phase was re-established (Fig. 4, C3 and C4). Like spiperone, NAD 299 caused a significant reduction in the half-width (Fig. 4D). Thus by blocking 5-HT receptors, the plateau-like oscillations are converted to the spike-like form, suggesting that 5-HT is endogenously released and causes a prolongation of the plateau phase. This modulatory action of 5-HT may primarily be mediated via 5-HT1A receptors. The converse experiment is illustrated in Fig. 5, where the control oscillations are of the spike-like form (Fig. 5, A1 and C1). After application of 5-HT, the oscillations were converted to the plateau-like form (Fig. 5, A2 and C2; n ⫽ 6). When spiperone was also applied, the oscillations again became spike like (Fig. 5A3), as was the case upon application of NAD 299 (Fig. 5C3). Application of 5-HT thus caused a significant prolongation of the half-width of the depolarized phase during 35 36 ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY We therefore tested whether the modulatory influence of 5-HT on the oscillations is exerted solely via an action on L-type calcium channels or if other routes of action may also contribute. Application of the L-type calcium channel antagonist nimodipine prolongs the plateau phase of the oscillations, as does 5-HT (Fig. 6, A and B) (Wang et al. 2013). In the presence of nimodipine, 5-HT was, however, still able to prolong the plateaus further (Fig. 6C; n ⫽ 3), suggesting that the action of 5-HT on NMDA-induced membrane potential oscillations is not only exerted via a blockade of L-type calcium channels (CaV 1.3). DISCUSSION A Control (150µM NMDA) -40 mV -60 5s B 10µM Nimodipine 17 min C 10µM Nimodipine + 1µM 5-HT 12 min Fig. 6. Blockade of L-type calcium channels does not occlude the effect of 5-HT application. A: control; spike-like membrane potential oscillations induced by bath application of 150 M NMDA in the presence of TTX. B: following application of the L-type calcium channel blocker nimodipine, oscillations became more plateau like (cf. Wang et al. 2013). C: in the presence of nimodipine, 5-HT application further prolonged the oscillation plateaus. Time and voltage calibrations in A apply to all traces. J Neurophysiol • doi:10.1152/jn.00582.2013 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 18, 2017 NMDA can induce pacemaker-like oscillations in different neurons in the lamprey spinal cord and in other species (Hochman et al. 1994; Li et al. 2010; Masino et al. 2012; Sigvardt et al. 1985; Wallén and Grillner 1987) after a blockade of interaction between the cells with tetrodotoxin. The generation of these plateaus depends on an interaction between a number of ion channels. The voltage dependence of the NMDA receptors is central to the oscillations, but the depolarization is counteracted by voltage-dependent potassium channels, and LVA calcium channels also contribute to the depolarization (Grillner and Wallén 1985; Wang et al. 2013). The termination of the plateau phase is due to activation of KCa channels and a closure of the voltage-dependent NMDA receptors (El Manira et al. 1994; Wallén and Grillner 1987). It has long been noted that these oscillations in the lamprey can either have a pronounced, depolarized plateau phase or show a continuous decrease of the amplitude after the peak without a significant plateau. Different spinal cord preparations have been found predominantly to express either of these two types of pacemaker-like oscillations. We have here first characterized the properties of these two versions. Both types of oscillations show a similar dependence on the level of NMDA receptor activation, indicating that the same ion channel mechanisms underlie both types [cf. Wallén and Grillner (1987); Wang et al. (2013)]. On the basis of these findings, we then set out to investigate whether differences in modulatory, serotonergic influence may explain the presence of the two types of oscillations in different preparations. We now show that this appears to be the case and that the spinal 5-HT system indeed may contribute to the shape of the oscillations. A related finding is that Zhang and Grillner (2000) have shown that during fictive locomotion, a blockade of 5-HT receptors led to marked acceleration of the frequency of locomotor activity, suggesting that there was a tonic release of 5-HT affecting the locomotor frequency. This is most likely caused by the intraspinal 5-HT neurons located below the central canal (Schotland et al. 1995; van Dongen et al. 1985; Zhang et al. 1996). The plateau-like oscillations induced by NMDA in the spinal cord preparation were transferred to the spike-like form of oscillations after a blockade of 5-HT receptors with spiperone [cf. Zhang and Grillner (2000)] and also with NAD 299, a specific 5-HT1A antagonist. This suggests that there is an endogenous release of 5-HT also during the oscillations. Furthermore, since this release is maintained in the presence of TTX, it may occur spontaneously in the absence of spikes. The release of 5-HT causes a depression of KCa channel activation (El Manira et al. 1994; Wallén et al. 1989) and thereby a prolongation of the plateau, and consequently, a blockade of 5-HT receptors will enhance the level of KCa channel activity, which will terminate the plateau-like oscillations earlier and make them spike like. Conversely, an enhanced level of 5-HT, as during external application in the present experiments, will depress KCa channel activation and thereby transform the spike-like oscillations to the more plateau-like version. Application of 5-HT is also known to slow down the locomotor frequency (Harris-Warrick and Cohen 1985). A modulation of TTX-resistant NMDA oscillations by 5-HT occurs in several species, including rodents (Hsiao et al. 2002; MacLean et al. 1998; MacLean and Schmidt 2001; Masino et al. 2012; Schmidt et al. 1998), amphibians (Reith and Sillar 1998; Scrymgeour-Wedderburn et al. 1997; Sillar and Simmers 1994a; Sillar et al. 1992), and lamprey [present study and El Manira et al. (1994); Wallén et al. (1989)]. 5-HT will reduce KCa channel activation through an action on voltage-dependent calcium channels (Hill et al. 2003). During NMDA-induced membrane potential oscillations, which range between approximately ⫺70 and ⫺40 mV, LVA calcium channels of the CaV 1.3 subtype contribute to KCa channel activation, in addition to calcium entry via other routes, notably NMDA receptor channels (Alpert and Alford 2013; Nanou et al. 2013; Wang et al. 2013). We have furthermore shown that this subtype of LVA calcium channel also contributes to the PIR response in lamprey spinal neurons and that 5-HT acts on the CaV 1.3 subtype to modulate the PIR (Wang et al. 2011). In the case of the 5-HT modulation of the NMDA-induced membrane potential oscillations studied here, the action appears not to be mediated only via CaV 1.3 subtype calcium channels, ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY ACKNOWLEDGMENTS We are grateful to Dr. Brita Robertson for valuable comments on the manuscript. GRANTS Support for this study was provided by the Swedish Research Council (Medicine, project no. 3026; Natural and Engineering Sciences, project no. 1496), the European Union (project nos. QLG3-CT-2001-01241 and HealthF2-2007-201144, FP7), and the Karolinska Institute Foundations. Support for the study was also provided by a grant from the National Natural Science Foundation of China (grant no. 31371198) and the Natural Science Foundation of Liaoning Province, People’s Republic of China (grant no. 201202144), and by grants from the Scientific Research Foundation for the Returned Overseas Chinese Scholars by the State Education Ministry and the Ministry of Human Resources and Social Security of the People’s Republic of China (grant no. 2011LX007), received by D. Wang. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. 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