A longitudinal and bilateral asymmetry of the serotonin function in the regulation of motor activity of the rabbit vas deferens in vivo
Alla E Lychkova (lychkova at mail dot ru) #, Alexader M Puzikov
Moscow’s Clinical Research Practical Center, Shosse Enthusiasts 86, 111123 Moscow, Russian Federation
# : corresponding author
DOI
//dx.doi.org/10.13070/rs.en.2.1475
Date
2015-12-16
Cite as
Research 2015;2:1475
License
Abstract

Introduction: Contractile responses of the rabbit vas deferens right and left branches to vagus nerve and serotonin (5-HT) stimulation were compared. Materials and Methods: The vas deferens electromyogram (EMG) was measured by Nihon Kohden, Neurofax, EEG 4400, using surface bipolar silver electrodes (contact area 1.5-2.0 mm2, distance between electrodes 1.5 mm) for extracellular recordings of slow waves frequency and amplitude. Results: Electric field stimulation of the vagus nerve markedly increased contraction of the right branch of the rabbit vas deferens compared with the left one. Further addition of serotonin (50 mg/kg) markedly increased the contraction in the right compared with the left branch of the rabbit vas deferens. Sumatriptan or droperidol administration abolished the contractile response in the rabbit vas deferens. Conclusion: These findings may indicate that contribution of cholinergic and serotonergic neurotransmission to the contractile response of the right branch rabbit vas deferens is different from that of the left one.

Introduction

The vas deferens (ductus deferens) is the least understood and studied organ of the male reproductive system [1]. The vas deferens is a paired (right and left) muscular duct that forms the distal extension of the ductus epididymidis and transmits spermatozoa and seminal fluids from the epididymis and seminal vesicles to the ejaculatory duct. The vas deferens also contributes to secretion of fluid for sperm transport and possibly to resorption of spermatozoan remnants from the duct lumen.

In cross section, the vas deferens consists of an outer adventitial connective tissue sheet containing blood vessels and small nerves, a muscular coat that in human consists of a middle circular layer surrounded by inner and outer longitudinal muscle layers, and an inner mucosal layer with an epithelial lining [2]. The vas deferens conditionally can be separated into three parts: the proximal vas deferens (epididimal), the medial vas deferens, and the distal vas deferens (prostatic) parts.

An important characteristic of the vas deferens is its contractile activity. The vas deferens is the most muscular of all the tubular organs in the body [3]. The rapid transport ability is consistent with the vas deferens having the greatest muscle to lumen ratio (≈10 : 1) of any hollow viscus in the body. The total thickness of the human vas ranged from 1.5 to 2.7 mm (mean, 1.89 mm); the lumen of the vas ranged from 0.2 to 0.7 mm (mean, 0.43 mm) [4].

Serotonin (5-HT) is the most important neurotransmitter in the control of ejaculation and its impact on ejaculation has been demonstrated in animal and human models [5]. 5-HT is a regulatory and biologically active neurotransmitter in the CNS and peripheral tissues [6, 7]. 5-HT is primarily produced by enterochromaffin cells of the intestine, and in the vas is stored into platelets, smooth muscles, and in mast cells.

In the last decade, more attention attract the participation of serotonin in the regulation of contractile activity of the vas deferens [8, 9]. In earlier paper we suggest that endogenous serotonin participate in the regulation of smooth muscle activity through potentiation of the excitatory action of acetylcholine in the vas deferens [10].

There are also plenty neuronal, hormonal, metabolic, and mechanical factors influencing contractile function of the vas. The motor innervation of the smooth muscle coat of the human vas deferens is predominantly adrenergic [11, 12] and adrenergic nerve fibers have been observed in all three layers of the vas tunica muscularis, while a less dense and differently distributed presumptive cholinergic innervation is also in evidence, although the precise role of the latter is undetermined [12-14], histaminergic [15]. The vas deferens receives innervation also from an abundant ligand-gated purinergic receptors in its smooth muscle membranes, suggesting sympathetic and purinergic cotransmission in sperm transport and ejaculation [16-19].

Then, considerable contribution of cholinergic innervation could not exclude in rabbit vas deferens, besides adrenergic and purinergic innervation [20] ; recently attention are increased to nitrergic [21, 22], and peptidergic nerve fibers [23, 24].

Serotonin and other drugs influence on contractile function of the vas deferens are mediated by corresponding receptors.

5-HT induce different mechanical responses (i.e, contraction, relaxation or both) on vascular and non-vascular smooth muscles (gastrointestinal tract, heart, urethra, prostate, uterus) of several mammalian species. The tissue- and species-related variations in 5-HT-induced mechanical responses are due to the multiple 5-HT receptor subtypes and their heterogeneous expression in the different organs [25, 26]. Seven groups (from 5-HT1 to 5-HT7) and several tens of serotonin receptor subtypes have been classified by International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification on the basis of their structural and signaling features [27]. With the exception of 5-HT3 receptor which is coupled with ion channels and is exclusively expressed in neural components, the other 5-HT subtypes are G-protein-coupled receptors). Human and rodent vas deferens smooth muscle cells express a following functional serotonin receptors: 5-HT1 in human [28], 5-HT1A subtype receptor in mouse [28], in rat [29, 30], 5-HT1B in rat [29], 5-HT2 in mouse [31], and in rat [32], 5-HT2C subtype in rat [29]. 5-HT2,3,4 expressed in the rabbit vas deferens [37] ; 5-HT7 receptors represented in the guinea pig [33].

Despite these findings, the mechanisms of serotonergic regulation of vas deferens parts are not fully understood.

The aim of the present study was to evaluate whether and how serotonin may enhance the contractile responses on acetylcholine of in vivo rabbit vas deferens and to determine longitudinal and bilateral asymmetry of serotonin contractile function in vas deferens.

Materials and methods
Animals

The electrophysiological experiments were performed on twelve male Chinchilla rabbits, weighing 2.5–3.5 kg, and 5,5–6,5 months of age. The animals were provided from the Animal Facility of the Russian National Research Medical University, Moscow, Russian Federation. Experiments were carried out in accordance with national ethical guidelines, and the animals were handled in a manner approved by the Institutional Animal Use and Care Committee of the Russian National Research Medical University.

Surgery

Animals were placed under the conditions of the surgical stage of Nembutal narcosis (40 mg/kg, intraperitoneally), and inferior-medial laparotomy was performed. Access to the vas deferens was opened. The paired electrodes were superimposed on the surface of the following parts of the vas deferens: the proximal, the middle, and the distal vas deferens parts in vivo. Contact between the electrode tips and the vas deferens surface was achieved. Control experiments confirming the absence of instrument-derived artifacts were carried out following standard procedures [Ballaro A, 2008).

Drugs

The drugs used in this study (obtained from the sources indicated) were: Droperidol (PubChem CID:3168) (Droleptan), used at the dose of 1.0 mg/kg body weight, was from Gedeon Richter Ltd. Sumatriptan succinate from Glaxo Group Research, Ware, UK. All drugs were dissolved in physiological 0,9% NaCl solution immediately before use.

Measurements of vas deferens EMG

The vas deferens EMG was measured using surface bipolar silver electrodes (contact area 1.5-2.0 mm2, distance between electrodes 1.5 mm) for extracellular recordings. EMG recording was performed with a 21-channel electroencephalograph (Nihon-Kohden, Neurofax, EEG 4400 series, Washington, DC).

Electrical stimulation of nerves

The ЕМ-42 Medicor (Hungary) electro stimulator was used to stimulate cholinergic and serotonergic nerve fibers (the last - as part of the sympathetic trunk). Electric field stimulation was applied to the peripheral cervical segment of the right vagus nerve. The level of parasympathetic nerve stimulation was sufficiently low (2 msec, 1.5-7.0 V, 10 Hz,), so that the vas deferens contraction rate remained stable during 60-90 s in each experiment. Electrical stimulation was also applied to the serotonergic fibers contained in the peripheral cervical segment of the left sympathetic trunk.

Morphological data

Strips of right vas deferens tissue were fixed in 9% formalin, dehydrated in alcohols of increasing concentration, embedded in paraffin, histologic sections were prepared, stained with hematoxylin and eosin.

Statistical analysis

Data are expressed as means ± standard error. Student’s t test was used for statistical comparisons when appropriate, and differences were considered significant at P < 0.05.

Results and discussion
Methodological approach

We had previously described and used an electrophysiological approach to evaluate in vivo the action of peripheral 5-HT on contractile activity of smooth muscles of different organs and tissues [35-38]. In this study, we extended this approach to the both vas deferens and its three parts, evaluating the effect of electric stimulation of sympathetic trunk (e.g, serotoninergic fibers in it) on EMG induced by parasympathetic nerve (the vagus nerve) stimulation in the distal (the prostatic), the middle, and the proximal (the epididimal) parts of the rabbit vas deferens.

The presence of 5-HT in the sympathetic trunk has been well ascertained by both direct and indirect (functional) methods. In earlier observation, Jaim-Etcheverry and Zieher (1980) indicated the presence of 5-HT in the fibers of the sympathetic trunk (the superior cervical ganglia) of the rat [39]. Smirnov and Volyntseva (1995) localized 5-HT in the sympathetic trunk of the rat using the Falck method, suggesting that the sympathetic trunk contains massive quantity of 5-HT in its fibers [40]. Later, Smirnov and co-workers confirmed the presence of 5-HT in the sympathetic trunk of pigeons, guinea pigs, rats, cats, rabbits, and dogs by functional test on gastric motility [41-43]. Thus, it appears reasonable to assume that activation of the rabbit sympathetic trunk leads to excitation of the serotonergic fibres and subsequent 5-HT release.

Drug EMG base level EMG after vagus
nerve stimulation
EMG after simultaneous
stimulation of the vagus
nerve and serotonin
administration
Frequency Amplitude Frequency Amplitude Frequency Amplitude
The right vas deferens proximal part
__14.0±1.70.17±0.0318.0±1.30.25±0.0321.0±1.70.33±0.05
droperidol6.0±0.30.27±0.028.3±0.70.33±0.056.8±0.60.27±0.05
sumatriptan6.0±0.30.25±0.049.5±0.40.25±0.049.5±0.50.2±0.02
5-HT15.3±0.70.17±0.0217.3±0.80.2±0.0322.7±1.20.23±0.03
The right vas deferens middle part
__9.3±1.80.2±0.0213.2±1.90.23±0.0518.5±2.30.27±0.05
droperidol9.0±0.80.2±0.0212.0±1.20.2±0.0212.0±1.50.19±0.03
sumatriptan9.0±1.50.18±0.0313.5±1.50.19±0.0411.0±1.00.17±0.03
5-HT6.3±0.30.2±0.0211.0±1.30.23±0.0217.6±3.80.23±0.03
The right vas deferens distal part
__9.8±0.80.23±0.0313.3±1.20.23±0.0318.0±1.70.26±0.05
droperidol8.0±1.00.15±0.0111.5±0.50.34±0.0611.0±0.80.16±0.05
sumatriptan7.1±0.50.25±0.039.5±1.10.25±0.038.0±0.70.25±0.04
5-HT10.2±0.80.2±0.0313.0±1.10.3±0.0416.3±1.40.5±0.01
Table 1. EMG prior nerve excitation, under vagus nerve stimulation and under simultaneous stimulation of the vagus nerve and sympathetic trunk of rabbit right vas deferens, before and after treatment with exogenous 5-HT, or 5-HT receptor inhibitors droperidol and sumatriptan, Frequency, /min; amplitude, mV. P<0.05 for all data reported.
The right vas deferens proximal part

The vagus nerve by itself cause the increasement of 28.5% of the EMG frequency and amplitude of 47% of slow wave of the proximal vas deferens (Table 1). The contraction is potentiated by stimulated the sympathetic trunk that added another 16.6% to the frequency value and additional 49% to amplitude value of EMG slow waves of the vas deferens (Table 1). Consequently, the addition of the stimulation of the sympathetic trunk to stimulation of the vagus nerve enhances vagal stimulatory influence on the smooth muscle EMG of the vas deferens proximal part.

In the next series of experiments, EMG frequency and amplitude of the right vas deferens proximal part contractions were measured before and after the intravenous (i.v.) administration of droperidol. The administration of this inhibitor associated with stimulation of the vagus nerve increased the frequency of the slow wave EMG by 38.3%, while the amplitude of the slow waves increased by 32.3% (Table 1). Droperidol administration together with jointly stimulation of the vagus nerve and the sympathetic trunk inhibited the effects: the frequency of EMG slow waves was 6.8±0.6 /min, amplitude 0.27±0.05 mV (Table 1). Consequently, in the implementation of the phenomenon are participated serotonergic neurons that express on the its surface membrane 5-HT3.4-receptors.

The study of 5-HT1,2 receptor role was made with the 5-HT1,2 receptor inhibitor sumatriptan. Sumatriptan administration increased both the EMG frequency of slow waves составила 6.0 ± 0.3 /min, and amplitude 0.25 ± 0.04 mV (Table 1). Until the introduction of sumatriptan - blocker of 5-НT1,2 receptors - stimulation of the sympathetic trunk in connection with the stimulation of the vagus nerve leads to increased frequency of slow waves of EMG with 16.0 ± 2.0 / min to 23.5 ± 3.5 / min (47%, p < 0.05 ), the amplitude was 0.2 ± 0.01 mV. Sumatriptan administration together with the jointly stimulation of the vagus nerve and of the sympathetic trunk inhibited the effect: the frequency decreases to 9.5 ± 0.5 /min (р > 0.1) and the amplitude of EMG slow waves 0.2 ± 0.02 мВ (Table 1).

The serotonergic nature of the sympathetic nerve stimulation was revealed by the results of experiments with the direct administration of serotonin (in a dose of 25 mkg / kg). The dose of serotonin was subliminal and independent influence 5-HT on electromotor activity of the vas deferens is absent. Stimulation of the vagus nerve in this series of experiments led to an increasement in the frequency of slow waves with EMG 15.3 ± 0.7 to 17.3 ± 0.8 min (13.1%, p <0.05) and with amplitude 0.17 ± 0.02 to 0.20±0.03 mV (17.6%, p< 0.05). Introduction of serotonin on the background of stimulation of the vagus nerve leads to a further increasement in the frequency of slow waves in the EMG to 22.7 ± 1.2 min (31.2%, p < 0.05) and amplitude - to 0.23 ± 0.03 mV (15.0%, p < 0.05). Consequently, the introduction of serotonin as sympathetic nerve stimulation, increases vagal stimulatory effect on the proximal part of the right vas deferens.

The right vas deferens middle part

Stimulation of the vagus nerve led to an increase of 42% of the EMG frequency and amplitude of 15% (р < 0.05) of slow wave of the right vas deferens middle part (Table I). The jointly stimulation of the vagus nerve and the sympathetic trunk increased vagal stimulatory effect on EMG slow waves of smooth muscle of the right vas deferens middle part: the frequency of slow waves increased further by 40.1% (р < 0.05) and amplitude further by 17% (р < 0.05) (Table 1). Consequently, the summation of the stimulation of the sympathetic trunk with stimulation of the vagus nerve enhances vagal stimulatory influence on the smooth muscle EMG of the vas deferens middle part.

In the next series of experiments, EMG frequency and amplitude associated with the vas deferens middle part contractions were measured before and after the intravenous (i.v.) administration of droperidol. The administration of this inhibitor associated with stimulation of the vagus nerve increased the frequency of the slow wave EMG by 33.4%, while the amplitude of the slow waves remained constant - 0.23 ± 0.04 mV (Table 1), but fully inhibited effect of jointly stimulation of the vagus nerve and the sympathetic trunk (Table 1). Consequently, in the implementation of the phenomenon are involved serotonergic neurons that express on its the surface membrane 5-HT3.4-receptors.

The study of possible role of 5-HT1.2 receptor in realization of 5-HT vagal stimulatory effect on right vas deferens middle part EMG was conducted using the 5-HT1.2 receptor inhibitor sumatriptan. Stimulation of the vagus nerve led to an increasement of 45% of the EMG frequency, amplitude of the right middle part of the vas deferens remain constant (Table 1). Joint stimulation of the sympathetic trunk and stimulation of the vagus nerve lead to increased frequency of slow waves EMG by 47% (p < 0.05). Sumatriptan administration together with the jointly stimulation of the vagus nerve and the sympathetic trunk inhibited the fenomen (Table 1). Consequently, in the implementation of the phenomenon (i.e, synergic effect of the stimulation of the sympathetic trunk which enhance the vagal stimulatory influence) 5-HT1.2-receptors are involved.

Confirmation of serotonergic amplification mechanism of vagal stimulation of the sympathetic nerve EMG of the proximal part of the right vas deferens was the results of experiments with the introduction of serotonin in a dose of 25 mkg / kg. Stimulation of the vagus nerve in this series of experiments led to an increasement in the frequency of slow waves EMG from 6.3 ± 0.3 to 11.0 ± 1.3 /min (74.6%, p<0.05) and with amplitude from 0.2 ± 0.02 to 0.23 ± 0.02 mV (15%, р < 0.05). Serotonin administration together with field stimulation of the vagus nerve further increased the frequency of slow waves in the EMG to 17.6 ± 3.8 /min (60%, p<0.05) and stable amplitude. Consequently, serotonin administration as sympathetic nerve stimulation, enhanced vagal stimulatory effect on the proximal part of the right vas deferens.

The right vas deferens distal part

Stimulation of the vagus nerve led to 35.7% (p < 0.05) increase of the slow wave EMG frequency; amplitude remain constant (Table 1). The additional stimulation of the sympathetic trunk increased the vagal stimulatory effect: the frequency and amplitude of the EMG slow waves increased further by 35.4% (p < 0.05) and by 13%, respectively (Table 1).

On the background of droperidol administration stimulation of the vagus nerve increased the frequency of slow waves of the distal right vas deferens from 8.0 ± 1.0 to 11.5 ± 0.5 (43.8%, p < 0.05) and an amplitude of 0.15 ± 0.01 mV to 0.34 ±0.06 (113%, p < 0.05 ).

Pharmacological analysis showed that droperidol inhibits the effect of jointly activation of the sympathetic trunk together with the vagus nerve on the vas deferens distal part EMG (Table 1).

Sumatriptan administration increased the stimulatory influence of the vagus nerve by 33.8% with stable amplitude is 0.25 ± 0.03 mV (Table 1). Sumatriptan administration together with jointly vagus nerve and sympathetic trunk stimulation inhibit the vagus induced 5-HT enhanced stimulatory effect (Table 1).

The direct proof of serotonergic nature of the sympathetic trunk enhancement of vagal stimulation of the EMG of the proximal part of the right vas deferens came from the results of experiments with direct exogenous serotonin administration (in the dose of 25 mkg / kg). Stimulation of the vagus nerve in this series of experiments led to an increasement in the frequency of slow waves EMG from 10.2 ± 0.8 to 13.0 ± 1.1 /min (27.5%, p <0.05) and with amplitude from 0.2 ± 0.03 to 0.3 ± 0.04 mV (50%, р < 0.05). Serotonin administration together with field stimulation of the vagus nerve leads to a further increasement in the frequency of slow waves in the EMG to 16.3 ± 1.4 /min (25.4%, p <0.05) and amplitude - to 0.5 ± 0.01 mV (66.7%, p <0.05). Consequently, serotonin administration as well as the sympathetic trunk nerve stimulation, increased vagal stimulatory effect on the proximal part of the right vas deferens.

Drug EMG base level EMG after pelvic
nerve stimulation
EMG after simultaneous
stimulation of the pelvic
nerve and serotonin
administration
Frequency Amplitude Frequency Amplitude Frequency Amplitude
The left vas deferens proximal part
__9.0±0.50.21±0.0312.7±0.70.26±0.0514.1±1.00.4±0.06
droperidol14.0±0.80.2±0.0320.0±1.70.25±0.0420.0±1.10.25±0.04
sumatriptan9.5±1.20.2±0.0412.5±1.50.2±0.0411.6±1.40.2±0.03
5-HT12.0±0.50.2±0.0315.0±0.80.27±0.0517.0±1.20.5±0.08
The left vas deferens middle part
__8.7±0.30.17±0.0212.4±0.60.26±0.0316.0±0.50.37±0.04
droperidol9.1±0.50.14±0.0312.0±0.30.22±0.0312.0±0.40.21±0.03
sumatriptan8.3±0.30.13±0.0211.8±0.40.17±0.0311.0±0.30.16±0.04
5-HT9.0±0.60.25±0.0313.0±1.10.36±0.0415.6±1.00.38±0.02
The left vas deferens distal part
__9.5±0.50.15±0.0315.5±1.30.16±0.0421.0±1.90.19±0.05
droperidol8.7±0.50.16±0.0410.7±0.40.18±0.0310.3±3.30.17±0.04
sumatriptan8.3±0.40.15±0.0210.5±0.30.17±0.0210.2±2.10.17±0.03
5-HT13.0±0.40.23±0.0320.0±0.50.25±0.0524.2±0.70.3±0.06
Table 2. EMG prior nerve excitation, under vagus nerve stimulation and under simultaneous stimulation of the vagus nerve and sympathetic trunk of rabbit left vas deferens, before and after treatment with exogenous 5-HT, or 5-HT receptor inhibitors droperidol and sumatriptan, Frequency, /min; amplitude, mV. P<0.05 for all data reported.
The left vas deferens proximal part

Stimulation of the vagus nerve led to an increasement of 41.3% (p <0.05) of the EMG frequency and amplitude of 23.8% (p <0.05) of slow wave of the proximal left vas deferens (Table 2). The jointly stimulation of the vagus nerve and the sympathetic trunk increased vagal stimulatory effect on EMG slow waves of smooth muscle of the proximal left vas deferens: the frequency of slow waves increased further by 15.7% (p < 0.05) and amplitude by 61.6% (p < 0.05) (Table 2). Consequently, the summation of the stimulation of the sympathetic trunk with stimulation of the vagus nerve enhances vagal stimulatory influence on the smooth muscle EMG of the left vas deferens proximal part.

In the next series of experiments, EMG frequency and amplitude associated with the vas deferens proximal part contractions were measured before and after the intravenous (i.v.) administration of droperidol. The administration of this inhibitor associated with stimulation of the vagus nerve increased the frequency of the slow wave EMG by 42.8% (p <0.05), while the amplitude of the slow waves increased by 33.3% (p <0.05) (Table 2). Droperidol administration together with jointly stimulation of the vagus nerve and the sympathetic trunk inhibited the effect: the frequency of EMG slow waves was 20.0 ± 1.1 /min, amplitude remained constant (Table 2). Consequently, in the implementation of the phenomenon are involved serotonergic neurons that express on its the surface membrane 5-HT3.4-receptors.

The study of 5-HT1,2 receptor was conducted using the 5-HT1,2 receptor inhibitor sumatriptan. Sumatriptan administration the EMG frequency of slow waves was 9.5±1.2 / min, and the amplitude 0.2±0.04 mV remain constant (Table 2). Stimulation of the vagus nerve on the background of the action of sumatriptan increases the frequency of slow waves by 31.6% with stable amplitude.

Sumatriptan administration together with the jointly stimulation of the vagus nerve and the sympathetic trunk inhibited the effect (Table 2).

Confirmation of serotonergic amplification mechanism of vagal stimulation of the sympathetic nerve EMG of the proximal part of the left vas deferens was the results of experiments with the introduction of serotonin in the dose of 25 mkg / kg. Stimulation of the vagus nerve in this series of experiments led to an increasement in the frequency of slow waves EMG from 12.0 ± 0.5 to 15.0 ± 0.8 /min (25%, p<0.05) and with amplitude from 0.2 ± 0.03 to 0.27 ± 0.05 mV (35%, р > 0.05). Introduction of serotonin on the background of stimulation of the vagus nerve leads to a further increasement in the frequency of slow waves in the EMG to 17.0 ±1.2 /min (13.3%, p < 0.05) and amplitude - to 0.5 ± 0.08 mV (85.2%, p <0.05). Consequently, the introduction of serotonin as sympathetic nerve stimulation, increases vagal stimulatory effect on the proximal part of the left vas deferens.

The left vas deferens middle part

Stimulation of the vagus nerve led to an increasement by 30.7% of the EMG frequency and amplitude of 35.7% of slow wave of the left vas deferens middle part (Table 2). The jointly stimulation of the vagus nerve and the sympathetic trunk increased vagal stimulatory effect on EMG slow waves of smooth muscle of the left vas deferens middle part: the frequency of slow waves increased further by 15.3% and amplitude further by 11.1% (Table 2). Consequently, the summation of the stimulation of the sympathetic trunk with stimulation of the vagus nerve enhanced vagal stimulatory influence on the smooth muscle EMG of the left vas deferens middle part.

In the next series of experiments, EMG frequency and amplitude associated with the left vas deferens contractions were measured before and after the intravenous (i.v.) administration of droperidol. The administration of this inhibitor associated with stimulation of the vagus nerve increased the frequency of the slow wave EMG by 26.0%, while the amplitude of the slow waves increased by 46.1% (Table 2), but fully inhibited effect of jointly stimulation of the vagus nerve and the sympathetic trunk (Table 2). Consequently, in the implementation of the phenomenon are involved serotonergic neurons that express on the its surface membrane 5-HT3.4-receptors.

The study of possible role of 5-HT1,2 receptor in realization of 5-HT vagal stimulatory effect on the left vas deferens middle part EMG was performed using the 5-HT1,2 receptor inhibitor sumatriptan. Stimulation of the vagus nerve led to an increasement of 17.9% of the EMG frequency, amplitude of the middle part of the left vas deferens led to an increasement by 12.5% (Table 2). Sumatriptan administration together with the jointly stimulation of the vagus nerve and the sympathetic trunk inhibited the fenomen (Table 2). Consequently, in the implementation of the phenomenon (i.e, the summation of the stimulation of the sympathetic trunk which enhance the vagal stimulatory influence) 5-HT1.2-receptors are involved.

Confirmation of serotonergic nature of mechanism of vagal stimulation by the stimulated sympathetic nerve of the proximal part of the left vas deferens was the results of experiments with the serotonin administration (25 mkg / kg). Stimulation of the vagus nerve in this series of experiments led to an increasement of the frequency of slow waves EMG from 9.0 ± 0.6 to 13.0 ± 1.4 /min (44.5%, p<0.05) and the amplitude from 0.25 ± 0.03 to 0.36 ± 0.04 mV (44%, р < 0.05). Serotonin administration on the background of stimulation of the vagus nerve leads to the further increasement in the frequency of slow waves in the EMG to 15.6 ± 1.0 /min (20%, p<0.05) and amplitude - to 0.38 ± 0.02 mV (6%, p > 0.05). Consequently, the introduction of serotonin, as was with sympathetic nerve stimulation, increased vagal stimulatory effect on the proximal part of the left vas deferens.

The left vas deferens distal part

Stimulation of the vagus nerve led to 63.2% and to 6.7% increase of the slow wave EMG frequency and amplitude, respectively (Table 2). The additional stimulation of the sympathetic trunk increased the vagal stimulatory effect: the frequency and amplitude of the EMG slow waves increased further by 35.5% (p < 0.05) and by 18.8% (p < 0.05), respectively (Table 2).

In the next series of experiments, EMG frequency and amplitude of the vas deferens distal part contractions were measured before and after the intravenous (i.v.) administration of droperidol. The administration of this inhibitor associated with stimulation of the vagus nerve increased the frequency of the slow wave EMG by 23.0% (p < 0.05), while the amplitude of the slow waves increased by 12.5% (p > 0.05) (Table 2), but fully inhibited effect of jointly stimulation of the vagus nerve and the sympathetic trunk (Table 2). Consequently, in the implementation of the phenomenon are involved serotonergic neurons that express on its the surface membrane 5-HT3.4-receptors.

Sumatriptan administration increased the stimulatory influence of the vagus nerve by 26.5% and 13.3% of the EMG slow wave frequency and amplitude, respectively, of the vas deferens distal part (Table 2). Sumatriptan administration together with jointly vagus nerve and sympathetic trunk stimulation did not lead to vagus enhancement of stimulatory effect (table 2): the frequency remained at a value of 10.2 ± 2.1 /min, and amplitude remained stable.

Acetylcholine (vagus nerve field stimulation)
The right branch of the vas deferens The left branch of the vas deferens
part of the vas deferens part of the vas deferens
proximalmiddledistalproximalMiddledistal
28.541.835.741.042.563.1
Serotonin (sympathetic trunk and vagus nerve shared field stimulation)
The right branch of the vas deferens The left branch of the vas deferens
part of the vas deferens part of the vas deferens
proximalmiddledistalproximalmiddledistal
33.439.934.011.929.028.9
Table 3. The distribution of the contractile function of serotonin and acetylcholine along the rabbit vas deferens length, right/left branches and different parts of the vas deferens, %.

Confirmation of serotonergic nature of mechanism of vagal stimulation by the sympathetic nerve in the proximal part of the left vas deferens was the results of experiments with the introduction of serotonin (in the dose of 25 mkg / kg). Stimulation of the vagus nerve in this series of experiments led to an increasement in the frequency of slow waves EMG from 13.0 ± 0.4 to 20.0 ± 0.5 /min (53.8%, p<0.05) and with amplitude 0.23 ± 0.03 to 0.25 ± 0.05 mV (6%, р > 0.05). Serotonin administration on the background of stimulation of the vagus nerve leads to the further increasement of the slow waves EMG frequency to 24.2 ± 0.7 /min (21%, p < 0.05) and amplitude - to 0.3 ± 0.06 mV (20%, p > 0.05). I.e, exogenous serotonin as well as the sympathetic nerve stimulation, increases vagal stimulatory effect on the proximal part of the left vas deferens.

Table 3 data show the values of the vagus (acetylcholine) contractile function and serotonin contractile influence along the vas deferens length. As can be seen from table 3, the maximum of a contractile function of acetylcholine is placed on the distal (prostatic) part of the left vas deferens. The maximum of a contractile function of serotonin falls on the middle part of the left rabbit vas deferens.

A longitudinal  and  bilateral asymmetry of the serotonin function in the regulation of motor activity of the rabbit vas deferens in vivo figure 1
Figure 1. Rabbit vas deferens. Stained with haematoxylin and eosin. x 120.
Morphological studies

A comparative morphological study of the tissues of the vas deferens of two groups of adult rabbits: control (Fig. 1) and with high level of serotonin (Fig. 2) was carried out. Haematoxylin and eosin staining showed a well-preserved morphology of the ductuli efferentes (Fig. 1). The typical epithelium of ductuli efferentes with alternating groups of columnar and cuboidal cells (ciliated and non-ciliated cells), and also with circular smooth muscle cell layer, was present.

The lumen of the vas deferens is lined with two-row epithelium covered with bordure. Epithelial cells show signs of secretory activity. The epithelium of the vas deferens lies on the basement membrane, which separates it from the lamina propria, consisting of connective tissue rich in elastic fibers (Fig. 1). The muscular coat is formed with circular beams of smooth muscle cells and elastic fibers. In the interior of the muscle coat lies plexus containing ganglion cells that are innervate the bundles of smooth muscle cells. Outside the vas deferens is covered with connective adventitia.

A longitudinal  and  bilateral asymmetry of the serotonin function in the regulation of motor activity of the rabbit vas deferens in vivo figure 2
Figure 2. Rabbit dilated vas deferens with high serotonin level. Stained with haematoxylin and eosin. x 120.

Serotonin induce a decline of height of the two-row of the vas deferens epithelium, possibly due to excessive activation of secretory activity. The epithelium of the vas deferens mucosa layer lies on the basement membrane, which separates it from the lamina propria; serotonin produce the detachment of epiteliocytes from the basement membrane. Venous vessel appeared dilated under serotonin influence (Fig. 2).

On the basis of comparative morphological study can be noted an enhancement of the secretory activity of the epithelial cells of the vas deferens under serotonin influence and a slight thinning of the seminiferous epithelium in the testis. In all investigated tissues serotonin induce vasodilation of vessels that is characteristic morphological feature influence of serotonin on vascular smooth muscle, the vas deferens including.

The results of electrophysiological and morphological studies suggest that serotonin enhance spermatogenesis and motility of the vas deferens.

Discussion

To our knowledge, this is the first report showing asymmetry of contractile reaction of the right and the left branches of the rabbit vas deferens in vivo. To compare the contractile activity of the vas deferens braches a method of electromyography (EMG) was used [44]. The EMG signal is a biomedical signal that measures electrical currents generated in muscles during its contraction representing neuromuscular activities. This signal is normally a function of time and is describable in terms of its amplitude, frequency and phase. The nervous system always controls the muscle activity (contraction/relaxation). Hence, the EMG signal is a complicated signal, which is controlled by the nervous system and is dependent on the anatomical and physiological properties of muscles [45]. The variant of method electromyography is a surface electromyography, a technique whereby voltage-measuring electrodes attached to the surface of the skin are used to detect and/or infer various phenomena relating to muscular contractions [46]. Unfortunately, a method electromyography found only limited application in the study of the vas deferens [47-52]. We made extensive use of the technique of surface electromyography in our studies of contractile activity of the rabbit heart, smooth hollow organs of the gastrointestinal, biliary, broncho-pulmonary and urogenital tracts. The study of the nervous regulation of contractile activity of the vas deferens was one of the last stages of that research program.

The results of this study show that: 1) motor asymmetry of the right vis the left branches of the vas deferens EMG induced by acetylcholine, serotonin, and their combination is existed; 2) the maximum of the acetylcholine induced EMG is located at the distal part of the left vas; 3) 5-HT effect on the vas contraction depends on the preliminary activation of the vagus nerve; 4) serotonin enhance the vagus nerve induced contraction of the vas deferens, 5) maximum of the serotonin induced EMG is located at the middle part of the right vas deferens ; 6) 5-HT enhancing effect on contraction is mediated by activation of 5-HT1,2 receptors expressed on the smooth muscle cells, and by activation of 5-HT3,4 receptors expressed on the ganglion neurons.

The last finding fits with the results of our mapping of the peripheral 5-HT contractile function in smooth muscles of hole muscular organs of the rodents [10, 55]. From this mapping efforts seems to emerge: in the uterine tubes, the uterus, the urinary bladder, and the vas deferens serotonin contractile function is mediated through 5-HT1,2,3,4 receptors. This conclusion is consistent with the previous findings of other authors, that 5-HT stimulates contraction of human and rodents vas deferens smooth muscle by these, and 5-HT7 receptors [28-31, 33, 53]. This local maximum of serotonin contractile function may be explained by endogenous 5-HT presence in the middle part of the right vas either bigger expression of serotonin receptors in the middle part of the right vas deferens. We hypothesize, that the middle part of the vas deferens accommodate a clusters of cells which, like the cells of Cajal, perform pacemaker function. Our hypothesis is consistent with data of Turunc T. et al, that the vas deferens contains Cajal-like cells [54].

Similar pacemakers cells we found in the cardia of the stomach, on the border of fundus and antrum, in the bulbar part of the duodenum, in the proximal cecum and in extrahepatic biliary tract [55-57]. Similar results were obtained in his study by M. Takaki [58].

In conclusion, our study in the first time provide evidence of asymmetry of 5-HT contractile functions in the right and left rabbit vas deferens in vivo and that 5-HT induced contraction had maximum at middle part of the right rabbit vas deferens. We confirm our earlier report that serotonin influence in the vas deferens is mediated through an activation of the 5-HT1,2 receptors of the effector cells, and 5-HT3,4 ganglionic receptors. There receptors and serotonergic fibers of a sympathetic trunk may constitute the fragment of the peripheral serotonergic system that innervate visceral organs and systems.

Declarations
Competing interests

The authors declare that they have no competing interests.

Author Contributions

All the above mentioned authors have contributed to this article from data collection to final drafting work. All authors also declare to have read and approved the final manuscript.

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