Presynaptic Serotonergic Modulation of Spontaneous and Miniature ...

ISSN 0022-0930, Journal of Evolutionary Biochemistry and Physiology, 2016, Vol. 52, No. 5, pp. 359—368. © Pleiades Publishing, Ltd., 2016. Original Russian Text © N.I. Kalinina, G.G. Kurchavyi, A.V. Zaitsev, N.P. Veselkin, 2016, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2016, Vol. 52, No. 5, pp. 328—336.

COMPARATIVE AND ONTOGENIC PHYSIOLOGY

Presynaptic Serotonergic Modulation of Spontaneous and Miniature Synaptic Activity in Frog Lumbar Motoneurons N. I. Kalininaa, G. G. Kurchavyia, A. V. Zaitseva, and N. P. Veselkina,b a Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences,

St. Petersburg, Russia b St. Petersburg State University, St. Petersburg, Russia

e-mail: [email protected] Received December 29, 2015

Abstract—The effects of serotonin (5-HT, 30 μM) on spontaneous and miniature synaptic activity in lumbar motoneurons from the isolated Rana ridibunda spinal cord were investigated using intracellular recording. 5-HT increased the frequency of spontaneous (sPSPs) and miniature postsynaptic potentials (mPSPs). The effect of 5-HT on different subpopulations of mPSPs was multidirectional: it increased the frequency of glutamatergic excitatory mPSPs by 18% and decreased the frequency of glycinergic inhibitory mPSPs by 28%, but had no effect on the frequency of GABAergic inhibitory mPSPs. The amplitude and kinetic parameters of any subpopulation of mPSPs did not change. The data obtained show that 5-HT regulates the probability of glutamate and glycine release from the presynaptic terminals ending at frog spinal motoneurons. 5-HT shifts the balance between synaptic excitation and inhibition in the spinal neural network toward excitation. Thus, 5-HT participates in control of motor output and provides its facilitation. DOI: 10.1134/S0022093016050045 Key words: motoneuron, spinal cord, mPSP, 5-HT. Abbreviations: AMPA—α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; D-AP5—D2-amino-pentanoic acid, EPSP—excitatory postsynaptic potential; IPSP—inhibitory postsynaptic potential; sPSP—spontaneous postsynaptic potential; mPSP—miniature postsynaptic potential; GABA—γ-aminobutyric acid; KA—kainic acid; MP—membrane potential; NMDA—N-methylD-aspartate; AP—action potential; TTX—tetrodotoxin; 5-HT—5-hydroxytryptamine (serotonin).

INTRODUCTION An endogenous monoamine serotonin (5-hydroxytryptamine, 5-HT) phylogenetically and ontogenetically is one of the most ancient neurotransmitters in vertebrates. It is widespread in the CNS being implicated in the regulation of major physiological functions, such as hormone

secretion, sleep–wake cycle, motor control, immunity, nociception [1, 2]. Serotonin is suggested to influence the recovery of spinal functions following spinal cord transection [3]. Serotonergic neurons are located in the nuclei of the raphe and reticular formation projecting nearly to all regions of the brain and spinal cord [4, 5]. In mammals and amphibians, the descending

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serotonergic fibers [6, 7] establish synaptic contacts directly on the somatodendritic membrane of lumbar motoneurons [8]. 5-HT participates in the regulation of two functional systems in the mammalian spinal cord, motor output control and nociception, by modulating both neural excitation thresholds and synaptic transmission [9]. 5-HT was shown to facilitate motor activity and inhibit sensory inputs [10–14]. 5-HT influences neural activity via 14 genetically, pharmacologically and functionally different 5-HT receptors which refer to 7 families, 5-HT1–5-HT7 [15, 16]. All 5-HT receptors are metabotropic except for one ionotropic receptor, 5-HT3. Information about the effect of 5-HT on mammalian spinal neurons is quite discrepant. 5-HT can both facilitate cationic currents via the activation of 5-HT2 receptors and inhibit the same currents via the activation of 5-HT1A/7 receptors [9, 17]. The mechanisms of pre- and postsynaptic modulation of neural activity by serotonin are also described in the spinal cord of lower vertebrates, specifically in cyclostomes [18], Xenopus laevis [19] and reptiles [20]. Presynaptically, 5-HT receptors can modulate the release of the major neurotransmitters, such as glutamate, GABA, acetylcholine, dopamine and noradrenaline [9, 15]. Data on the presynaptic effect of 5-HT in the mammalian spinal cord concern mainly the neurons in the posterior horn [19, 21]. Since the presynaptic effect of serotonin in frog spinal motoneurons is practically unexplored, this study aimed to disclose the role of serotonin in controlling motor output of the frog spinal cord as well as to determine the modulating effect of 5-HT on synaptic inputs in spinal motoneurons of different ergicity. MATERIALS AND METHODS Experiments were conducted on the isolated spinal cord preparation of the frog Rana ridibunda. Dorsal laminectomy was performed under ether anesthesia. After removing the meninges, segments IX and X were isolated together with the roots as frontal sections 2–3 mm thick. One of them was fixed the rostral surface up in an experimental chamber. The preparative procedure, chamber layout and experimental design were de-

scribed in detail previously [22]. In experiments, sagittal sections of the same segments were used. The perfusion solution containing (mM) 100 NaCl, 2 KCl, 0.5 MgCl, 5.5 glucose, 1.5 CaCl2, 9 NaHCO3 and 2 Tris (pH 7.4–7.6) was aerated with a gas mixture (98% O2 and 2% CO2) at a temperature of 16–18 °C. The flow rate was 6 ml/min, and the bath volume was 0.5 ml. Potentials were recorded intracellularly from motoneurons in segments IX or X using glass microelectrodes with a tip diameter of 1–1.5 μm and resistance of 10–20 Mom, filled with 3M KCl. Motoneurons were identified by an antidromic AP evoked upon stimulation of the ventral root. Potentials were registered using a microelectrode differential amplifier with a bandwidth up to 5 kHz devised in our laboratory (B.T. Ryabov), digitalized at a frequency of 10–20 kHz using an ADC NI USB-6211 (National Instruments, USA), and recorded using a WinWCP computer program (Strathclyde Electrophysiology Software, UK). The background spontaneous postsynaptic activity (sPSP) was recorded in the normal physiological saline under the absence of synaptic input stimulation. To record the fraction of miniature postsynaptic potentials (mPSPs), TTX (1 μM), a sodium channel blocker, was added to the perfusion solution to suppress spike activity. To pharmacologically isolate the inhibitory fraction (mIPSP), the perfusion solution was added in the presence of TTX with kinurenate (1 mM) or CNQX (20 μM) and D-AP5 (40 μM). GABAergic and glycinergic mIPSPs were then isolated by the addition either of strychnine (2 μM) or gabasin (20 μM), respectively. Glutamatergic AMPA mEPSPs were recorded in the presence of TTX, D-AP5, strychnine and gabasin. Fresh 5-HT solution (30 μM) was added to the perfusion solution. All the reagents were purchased from Sigma-Aldrich or Tocris Bioscience. Spontaneous/miniature PSPs were recorded during 75 s. In search, the events exceeding the noise by 1.5–2 times (more than 150 μV) were sorted out. A search for mPSPs was performed using a Clampfit 10.2 software (Axon Instruments, USA) on the basis of a generated template response. From the events selected automatically the erroneously included noises were manually excluded. To analyze the mPSP parameters, from

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Fig. 1. Spontaneous and miniature PSP in motoneurons. (A): Records exemplifying spontaneous postsynaptic potentials (sPSPs) in normal perfusion solution (control); (B): records exemplifying miniature postsynaptic potentials (mPSPs) after addition to perfusion solution of a sodium channel blocker, TTX (1 μM). Asteriscs mark mPSPs of different ergicities selected for further analysis. Time base 1 s; (C): columnar diagrams showing changes in the sPSP and mPSP frequencies (white circles and white triangles—separate measurements); (D): same as in (B) for sPSP and mPSP amplitudes. Calibration: 1 mV, 100 ms.

75 to 1900 synaptic events were averaged. Analyzed were the event frequency, peak amplitude, rise time (in the range from 10 to 90% of the peak amplitude) and decay time (from 90 to 10% of the peak amplitude). For statistics and graphing, Sigma Plot 11.0 and MS Excel programs were used. The mean values were compared using the Student’s t-test. The data were presented as mean ± SEM.

RESULTS Synaptic activity was registered in 32 motoneurons with the stable resting MP from –65 to –80 mV. Characterization of spontaneous and miniature PSPs. In control, the sPSP frequency varied in different cells from 7.6 to 22.4 events/s, averaging 13.1 ± 2.4 (n = 6); the mean peak amplitude of


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Average parameters of sPSP and mPSP in different fractions under normal conditions and after 5-HT application Control Fraction

peak ampl., μV

rise time, ms

decay time, ms

frequency, events/s

N

peak ampl., μV

rise time, ms

decay time, ms

frequency, events/s

N

Spontaneous (sPSPs)

533 ± 114

2.6 ± 0.2

14.8 ± 1.2

13.1 ± 2.4

6

979 ± 233

2.4 ± 0.2

12.9 ± 1.6

24.2 ± 2.7*

5

Miniature (mPSP)

427 ± 33

2.8 ± 0.8

11.2 ± 1.3

8.0 ± 0.6

12

450 ± 23

2.5 ± 0.2

14.1 ± 1.5

10.2 ± 0.9*

7

Inhibitory (mIPSP)

399 ± 30

3.0 ± 0.3

15.2 ± 0.8

5.7 ± 0.4

4

360 ± 71

2.9 ± 0.6

13.6 ± 1.0

4.4 ± 0.3*

4

Glutamatergic (AMPA mPSP)

396 ± 29

2.5 ± 0.07

8.4 ± 0.4

3.4 ± 1.5

5

430 ± 12

2.3 ± 0.05

9.5 ± 0.34

4.0 ± 1.6*

5

Glycinergic (GLY mPSP)

448 ± 15

2.6 ± 0.1

10.6 ± 0.8

3.9 ±1.1

5

424 ± 17

2.4 ± 0.1

11.3 ± 0.8

2.8 ± 0.4*

5

GABAergic (GABA mPSP)

287 ± 29

3.9 ± 0.2

21.7 ± 1.1

1.5 ± 0.4

4

307 ± 23

3.3 ± 0.54

20.2 ± 2.3

1.4 ± 0.3

4

Mean ± SEM, * Statistically significant changes, p < 0.05 (Student’s paired t-test).

potentials was 533 ± 114 μV. In the presence of TTX (1 μM), which blocks APs in the preparation, the frequency of miniature synaptic events was lower than the sPSP frequency, averaging 8.0 ± 0.6 events/s (n = 12, p < 0.05). The mPSP amplitude was lower than the sPSP frequency making 427 ± 33 μV (n = 12, p < 0.05) (Fig. 1, see Table). Since mPSPs in frog spinal motoneurons are presented by synaptic events of three ergicities (glutamate-, glycin- and GABAergic), we studied the characteristics of each of the fractions (Figs. 2A, 2B, 2C). The events of each ergicity were isolated pharmacologically (see Materials and Methods). The frequency of GABAergic mIPSPs was lower (1.5 ± 0.4 events/s, n = 4) than that of glycinergic mIPSPs (3.9 ± 1.1 events/s, n = 5, p < 0.05) and glutamatergic mEPSPs (3.4 ± 1.5 events/s, n = 5; Fig. 2). We also compared the total frequency of inhibitory (glycinergic + GABAergic) and excitatory events and found out that the frequency of inhibitory mPSPs (5.7 ± 0.4 events/s, n = 4) is significantly higher than that of excitatory mPSPs (3.4 ± 1.5 events/s, n = 5, p < 0.05). This suggests a prevalence of spontaneous inhibitory influences in frog motoneurons. The portion of glutamatergic mEPSPs in the total mPSP population was about 42%, while glycinergic mIPSPs made up 49% and GABAergic

mIPSPs—19% (Fig. 2E). Thus, the experimentally obtained total of the pharmacologically isolated mPSP fractions (110%) exceeds the theoretical by 10% (see Discussion). Further, we analyzed the amplitude and kinetics of miniature events of different ergicities. It is important to note that both inhibitory and excitatory mPSPs in our experimental conditions at the resting MP are depolarizing. This can be explained by an increased intracellular Cl– concentration due to diffusion of these ions from the microelectrode and by a resultant shift in the equilibrium potential which in frogs approximates the MP [22, 23]. The amplitude of GABAergic mIPSPs was minimal (287 ± 29 μV, n = 4) and significantly different from that of glycinergic mIPSPs (448 ± 15 μV, n = 5, p < 0.05) and glutamatergic mEPSPs (396 ± 29 μV, n = 5, p < 0.05). The mPSP amplitudes in the latter two fractions were indistinguishable from one another (p > 0.05). mEPSPs and glycinergic mIPSPs shared the similar kinetic characteristics (rise and decay time), whereas both rise and decay time for GABAergic mIPSPs significantly (by 1.5–2 times) exceeded those for mEPSPs and glycinergic mIPSPs (see Table and Fig. 2D). Effect of 5-HT on total spontaneous activity. As soon as 2–3 min after addition of serotonin (5-



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Fig. 2. Ratio between glycinergic, glutamatergic and GABAergic fractions in the whole population of miniature postsynaptic potentials. (A): Pharmacologically isolated glycinergic mPSP fraction. (a) Glycinergic mPSPs recorded during perfusion with solution containing TTX (1 μM), D-AP5 (40 μM), CNQX (20 μM) and gabasin (20 μM); (b) after addition of strychnine (2 μM); (B): pharmacologically isolated glutamatergic (AMPA) mPSP fraction. (a) AMPA mPSPs recorded during perfusion with solution containing TTX (1 μM), D-AP5 (40 μM), strychnine (2 μM) and gabasin (20 μM); (b) after addition of CNQX (20 μM); C: pharmacologically isolated GABAergic mPSP fraction. (a) GABA mPSPs recorded during perfusion with solution containing TTX (1 μM), D-AP5 (40 μM), CNQX (20 μM) and strychnine (2 μM); (b) after addition of gabasin (20 μM); (D): averaged mPSPs of different ergicities; (a) averaged GLY mIPSPs (n = 113), (b) averaged AMPA mEPSPs (n = 137), (c) averaged GABA mIPSPs (n = 75); (E): columnar diagrams showing the contribution of glutamatergic, GABAergic and glycinergic mPSP fractions to the whole population of miniature postsynaptic potentials. Calibration: 0.8 mV; 0.5 s (for (A) and (B)), 0.5 mV; 0.5 s (for (C)), 200 μV; 20 ms (for (D)).

HT, 30 μM) to the perfusion solution, the sPSP frequency increased by 85% from 13.1 ± 2.4 to 24.2 ± 2.7 events/s (n = 5, p < 0.05), while the sPSP amplitude rose by 84% from 533 ± 114 to 979 ± 233 μV (n = 5, p < 0.05) (Fig. 3). The effect of serotonin was reversible, since after washing in the normal solution both frequency and amplitude of sPSPs reverted to the initial level in 20–30 min.

Effect of 5-HT on mPSP. Changes in the frequency and amplitude of sPSPs under the effect of 5-HT may to a large extent be due to an increase in the number of spontaneous APs in the preparation. Hence, to study the modulating effect of 5-HT on synaptic transmission, all further experiments were conducted in the presence of TTX (1 μM). While in the normal solution serotonin (30 μM) evoked


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Fig. 3. Effect of serotonin on sPSP. (A): Examples of records of spontaneous synaptic potentials (sPSPs) in normal perfusion solution (control); (B): sPSPs after addition of 5-HT (30 μM) to perfusion solution; (C): washing in normal solution; (D), (E): columnar diagrams showing increased sPSP frequency (D) and amplitude (E) in control and after addition of 5-HT (white circles and white triangles—separate measurements).

a motoneuron membrane depolarization of about 2–4 mV, under the TTX-induced block of spike activity this depolarization virtually disappeared. The frequency of all mPSPs increased under the effect of 5-HT by 28% from 8.0 ± 0.6 (n = 12) to 10.2 ± 0.9 events/s (n = 7, p < 0.05; Fig. 4A). It should be noted that the effect of 5-HT on the frequency of synaptic events of different ergicity proved to be multidirectional. We found an increase in the mEPSP frequency by 18% from 3.4 ± 1.5 (in control) to 4.0 ± 1.6 events/s (n = 5, p > 0.05) and a decrease in the frequency of glycinergic mIPSPs by

28% from 3.9 ± 1.1 to 2.8 ± 0.4 events/s (n = 5, p < 0.05). The frequency of GABAergic mIPSPs was almost invariable. We also tested the effect of 5-HT on all inhibitory events (glycine- + GABAergic) and found out that the mIPSP frequency decreased by 23% from 5.7 ± 0.4 to 4.4 ± 0.3 events/s (n = 4, p < 0.05; Fig. 4B). While under control conditions the frequency ratio of excitatory and inhibitory events was 3.4/5.7 = 0.60, under the effect of 5-HT this ratio increased up to 4.0/4.4 = 0.91. Thus, the prevalence of spontaneous inhibitory influences on spinal motoneurons disappeared.



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Fig. 4. Modulating effect of serotonin on frequency and amplitude of whole mPSP population and inhibitory mPSP fraction. (A): (a) control, mPSP under perfusion with solution containing TTX (1 μM); (b) increased mPSP frequency after addition of 5-HT (30 μM); (B): (a) control, inhibitory mPSP fraction (mIPSP) under perfusion with solution containing TTX (1 μM), D-AP5 (40 μM) and CNQX (20 μM); (b) decreased mIPSP frequency after addition of 5-HT (30 μM); (C), (D): columnar diagrams showing changes in frequency and amplitude of mPSP and sIPSP, respectively, before and against the background of 5-HT application (30 μM). Asteriscs (*) mark statistically significant changes in frequency (Student’s t-test, p < 0.05).

5-HT had practically no influence on the kinetic parameters (rise and decay time) and the amplitude of all mPSP types (Fig. 5, see Table). These findings suggest that serotonergic modulation of the miniature synaptic activity in motoneurons occurs mainly presynaptically. DISCUSSION In the present study we investigated the characteristics of miniature events of different ergicity in frog spinal motoneurons. The GABAergic events were shown to be characterized by a slower kinetics and lower amplitude compared to the glycinergic events, and this is well consistent with previous data [24]. At the same time, the kinetic and amplitude characteristics of the glutamatergic events are similar to the glycinergic.

We determined a quantitative frequency ratio of glutamatergic, glycinergic and GABAergic mPSPs, which was 42, 49 and 19%, respectively. The total frequency of all three mPSP fractions exceeded the frequency of the whole mPSP population by 10%, and it is likely to be explained by the presence of mixed GABA/glycine mIPSPs [24, 25] which were in this case considered twice. We also investigated the effect of serotonin on spontaneous and miniature events. Serotonin increased the frequency and amplitude of spontaneous events almost twofold, probably due to an increased number of spontaneous APs in frog spinal motoneurons. In the presence of TTX, serotonin significantly changed the mPSP frequency only by 28% and had no appreciable effect on the mPSP amplitude and kinetic parameters, indicative of its presynaptic action.


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Fig. 5. Modulating influence of serotonin on frequency and amplitude of glutamatergic, glycinergic and GABAergic mPSP fractions. (A): (a) Gglutamatergic (AMPA) mPSP fraction (control); (b) after 5-HT (30 μM) application; (B): (a) glycinergic mIPSP fraction (control); (b) decreased mIPSP frequency after addition of 5-HT (30 μM); (C), (D): columnar diagrams showing changes in frequency and amplitude of different mPSP fractions. Asteriscs (*) mark statistically significant changes in frequency (Student’s t-test, p < 0.05).

Our results demonstrated that serotonin influenced different mPSP fraction unequally. 5-HT increased the frequency of the glutamatergic mEPSP fraction and decreased that of the inhibitory glycinergic fraction, exerting no effect on the frequency of the GABAergic mIPSP fraction. The similar experiments on superficial dorsal horn neurons in the young rat spinal cord yielded the opposite results: 5-HT decreased the frequency of glutamatergic mEPSPs via the activation of 5-HT1A receptors [26] and increased the frequency of inhibitory (GABA and glycine) mIPSPs [21]. The dorsal horn of the spinal cord is known to accommodate the nociceptive pathway, and 5-HT exerts there an analgesic effect. In our experiments, 5-HT increased the frequency of glu-

tamatergic mEPSPs and reduced the frequency of glycinergic mIPSPs in motoneurons, well in line with the hypothesis by Jacobs and Fornal [27] that 5-HT inhibits sensory systems and excites motor activity. Serotonin-induced increase in the frequency of glutamatergic mEPSPs was described in excitatory neurons of the substantia gelatinosa [21]. In this study it was revealed that 5-HT decreases the frequency of glycinergic mIPSPs, suggesting a decreased probability of vesicular release of glycine from presynaptic terminals, however, the exact mechanism of its action in frog spinal motoneurons is still to be elucidated. 5-HT1B receptor-mediated presynaptic inhibition of the evoked glycinergic currents was documented in hypoglos-



sal motoneurons of neonatal rats. Activation of 5-HT1B receptors reduced the frequency of spontaneous miniature IPSPs while unaffecting their amplitude. From this, a presynaptic mechanism of inhibition mediated by 5-HT1B receptors was inferred [28]. The presynaptic mechanisms of 5-HT effect modulation were explored in giant nerve terminals (calyx of Held) of neonate rats. Direct recordings of calcium currents revealed that 5-HT reduced (inhibited) presynaptic potential-driven calcium currents but did not influence potentialdependent currents of potassium channels [29], while the presynaptic inhibitory effect of 5-HT was detectable during the early postnatal development (first 5 days) and almost disappeared at the later developmental stages. Thus, it was shown that potential-dependent calcium channels represent a major target for the presynaptic inhibitory effect of 5-HT.

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CONCLUSION The obtained results suggest the implication of serotonin receptors in the mechanism of presynaptic modulation of spontaneous vesicular release of glutamate and glycine from presynaptic terminals. Both an increase in the frequency of glutamatergic mEPSPs and a decrease in the frequency of glycinergic mIPSPs shift the balance between synaptic excitation and inhibition in the spinal neural network toward excitation, providing thereby the regulation of motor output. Further experiments with the use of specific agonists and antagonists are needed to identify specific types of serotonin receptors involved in the modulation of spontaneous synaptic activity in frog spinal motoneurons. Supported by the Program of the Presidium of the Russian Academy of Sciences no. 19 and the RFBR grant no. 15-04-05782.

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