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. 2016 Apr 28;594(15):4339–4350. doi: 10.1113/JP272073

Stimulation of dopamine D2‐like receptors in the lumbosacral defaecation centre causes propulsive colorectal contractions in rats

Kiyotada Naitou 1, Hiroyuki Nakamori 1, Takahiko Shiina 1, Azusa Ikeda 1, Yuuta Nozue 1, Yuuki Sano 1, Takuya Yokoyama 2, Yoshio Yamamoto 2, Akihiro Yamada 3, Nozomi Akimoto 3, Hidemasa Furue 3, Yasutake Shimizu 1,✉
PMCID: PMC4967751  PMID: 26999074

Abstract

Key points

  • The pathophysiological roles of the CNS in bowel dysfunction in patients with irritable bowel syndrome and Parkinson's disease remain obscure.

  • In the present study, we demonstrate that dopamine in the lumbosacral defaecation centre causes strong propulsive motility of the colorectum.

  • The effect of dopamine is a result of activation of sacral parasympathetic preganglionic neurons via D2‐like dopamine receptors.

  • Considering that dopamine is a neurotransmitter of descending pain inhibitory pathways, our results highlight the novel concept that descending pain inhibitory pathways control not only pain, but also the defaecation reflex.

  • In addition, severe constipation in patients with Parkinson's disease can be explained by reduced parasympathetic outflow as a result of a loss of the effect of dopaminergic neurons.

Abstract

We have recently demonstrated that intrathecally injected noradrenaline caused propulsive contractions of the colorectum. We hypothesized that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. Because dopamine is one of the major neurotransmitters of descending pain inhibitory pathways in the spinal cord, we examined the effects of the intrathecal application of dopamine to the spinal defaecation centre on colorectal motility. Colorectal intraluminal pressure and expelled volume were recorded in vivo in anaesthetized rats. Slice patch clamp and immunohistochemistry were used to confirm the existence of dopamine‐sensitive neurons in the sacral parasympathetic nuclei. Intrathecal application of dopamine into the L6–S1 spinal cord, where the lumbosacral defaecation centre is located, caused propulsive contractions of the colorectum. Inactivation of spinal neurons using TTX blocked the effect of dopamine. Although thoracic spinal transection had no effect on the enhancement of colorectal motility by intrathecal dopamine, the severing of the pelvic nerves abolished the enhanced motility. Pharmacological experiments revealed that the effect of dopamine is mediated primarily by D2‐like dopamine receptors. Neurons labelled with retrograde dye injected at the colorectum showed an inward current in response to dopamine in slice patch clamp recordings. Furthermore, immunohistochemical analysis revealed that neurons immunoreactive to choline acetyltransferase express D2‐like dopamine receptors. Taken together, our findings demonstrate that dopamine activates sacral parasympathetic preganglionic neurons via D2‐like dopamine receptors and causes propulsive motility of the colorectum in rats. The present study supports the hypothesis that descending pain inhibitory pathways regulate defaecation reflexes.

Key points

  • The pathophysiological roles of the CNS in bowel dysfunction in patients with irritable bowel syndrome and Parkinson's disease remain obscure.

  • In the present study, we demonstrate that dopamine in the lumbosacral defaecation centre causes strong propulsive motility of the colorectum.

  • The effect of dopamine is a result of activation of sacral parasympathetic preganglionic neurons via D2‐like dopamine receptors.

  • Considering that dopamine is a neurotransmitter of descending pain inhibitory pathways, our results highlight the novel concept that descending pain inhibitory pathways control not only pain, but also the defaecation reflex.

  • In addition, severe constipation in patients with Parkinson's disease can be explained by reduced parasympathetic outflow as a result of a loss of the effect of dopaminergic neurons.

Abbreviations

ChAT

choline acetyltransferase

D2R

dopamine D2 receptor

ENS

enteric nervous system

FD

fast Dil

IML

intermediolateral cell column

PBS

phosphate‐buffered saline

PD

Parkinson's disease

SPN

sacral parasympathetic nuclei

Introduction

Dopamine is an important monoamine neurotransmitter in the CNS that is involved in the regulation of pain, the reward system, emotion and cognition (Nieoullon, 2002; Wise, 2004; Chinta & Andersen, 2005; Carlino & Benedetti, 2016). Dopamine exerts these actions via receptors classified into two families: the D1‐like and D2‐like receptors (Missale et al. 1998; Vallone et al. 2000; Alexander et al. 2015). Stimulation of D1‐like receptors results, via Gs, in stimulation of adenylate cyclase. By contrast, stimulation of D2‐like receptors mainly leads, via Gi/o, to inhibition of the enzyme activity (Millan, 2002). Therefore, dopamine can exert excitatory and inhibitory effects on neuronal activity. In addition to its central actions, dopamine has peripheral roles in the regulation of gastrointestinal motility. Gastrointestinal motility is controlled not only by the CNS, but also the enteric nervous system (ENS), and dopamine receptors are expressed in a subset of neurons of the ENS. As a result, dopamine has various action sites yielding various effects. It has been reported that peripheral dopamine inhibits gastrointestinal motility (Li et al. 2006; Zhang et al. 2012). By contrast to the peripheral action, agonists for D1‐ and D2‐like receptors enhance colonic motility when injected intracerebroventricularly (Bueno et al. 1992). Furthermore, constipation has been identified as a frequent symptom in patients with Parkinson's disease (PD) (Jost, 2010; Sakakibara et al. 2011). Considering the fact that PD is characterized by degeneration of dopaminergic neurons, dopamine is an important neurotransmitter mediating the defaecation reflex.

Although most parts of gastrointestinal tract receive extrinsic innervation by vagal nerves, the distal colon and rectum are dominantly innervated by parasympathetic pelvic nerves. The nerves arise from the lumbosacral defaecation centre located at the level of the L6–S1 spinal cord, and the spinal defaecation centre interacts with the defaecation centre in the brain stem. We have recently demonstrated that intrathecal injection of the monoamine neurotransmitter noradrenaline caused propulsive contractions of the colorectum in rats (Naitou et al. 2015 b). In the spinal cord, noradrenaline derived from the brain modulates pain signal transmission, referred to as descending pain inhibition (Pertovaara, 2006). Based on these facts, we hypothesized that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. This hypothesis is reasonable considering that the excretion of intracolonic contents relieves pain under physiological conditions.

It has been established that dopamine, as well as noradrenaline, is involved in descending pain inhibition (Millan, 2002). Recently, Karasawa et al. (2014) reported that denervation of brain dopaminergic neurons using microinjection of 6‐hydroxydopamine into the median forebrain bundle decreased c‐Fos expression in the intermediolateral cell column (IML) of the lumbosacral spinal cord (Karasawa et al. 2014). Taken together, these observations allow us to propose the hypothesis that dopaminergic neurons projecting to the spinal defaecation centre have a role in controlling colorectal motility. In the present study, we investigated whether dopamine injected into the lumbosacral spinal defaecation centre affects colorectal motility using an in vivo experimental system in anaesthetized rats (Bogeski et al. 2005; Shimizu et al. 2006). We also investigated the site of dopamine action in the spinal defaecation centre using immnohistology and the patch clamp method. The results obtained show that intrathecally injected dopamine caused propulsive contractions of the colorectum by activating sacral parasympathetic preganglionic neurons via D2‐like dopamine receptors. To our knowledge, this is the first study showing that the spinal dopamine system is involved in controlling the defaecation reflex.

Methods

Ethical approval

The experimental procedures were performed in accordance with the guidelines for the care and use of laboratory animals approved by the Animal Care and Use Committee of Gifu University.

Animals

Male Sprague–Dawley rats (Japan SLC, Inc., Shizuoka, Japan) were used. The rats were maintained in plastic cages under a 12:12 h light:dark cycle (lights on 06.00 h) at 22°C and were supplied with both laboratory chow (MF, Oriental Yeast Co.,Ltd, Tokyo, Japan) and water ad libitum prior to the experiments.

Recording of colorectal motility

The procedures for recording colorectal motility were based on those described previously (Bogeski et al. 2005; Shimizu et al. 2006). Rats (8–10 weeks old) were sedated with ketamine hydrochloride (50 mg kg−1, i.m. injection) and anaesthetized with α‐chloralose (60 mg kg−1, into the tail vein). The femoral artery was cannulated and anaesthesia was maintained by intra‐arterial infusion of α‐chloralose (10–20 mg kg−1 h−1) combined with ketamine hydrochloride (3–5 mg kg−1 h−1) in 0.9% saline. The arterial cannula was connected to a pressure transducer for monitoring of arterial blood pressure. Body temperature was maintained at 36–37ºC using a heating lamp throughout the experiments. On completion of the experiments, rats were immediately killed by i.p. injection of a lethal dose of sodium pentobarbitone (100 mg kg−1) when they were still under anaesthesia.

The colorectum of each anaesthetized rat was cannulated and tied around both in the region of the distal colon behind the bladder and at the anus, and then the body wall was closed around the oral cannula. The oral cannula was connected to a Mariotte bottle filled with warm saline kept at 37°C and the aboral cannula was connected to a pressure transducer and a fluid outlet through a one‐way valve for measurements of intraluminal pressure of the colorectum and expelled fluid volume, respectively. The basal level of intraluminal pressure was maintained at 3–5 mmHg by adjusting the heights of the Mariotte bottle and the outlet tube. Expelled fluid from the aboral cannula was collected in a cylinder positioned beneath the fluid outlet and measured with a force transducer.

When the spinal cord was transected at T8 during the recording session, the region of the T8 vertebra was exposed by laminectomy and the spinal cord was transected with microscissors (Shima et al. 2014). In some series of experiments, the parasympathetic pelvic nerves were bilaterally cut before the colorectal cannulation (Hirayama et al. 2010). After the surgical operation for recording of colorectal motility, rats were kept for ∼1 h to allow basal colorectal motility and stabilization of blood pressure.

For application of drugs to the lumbosacral spinal cord, a 30‐gauge needle connected to a polyethylene tube was inserted between the L1 and L2 vertebrae from the dorsal surface, until tail flick appeared (L1–L2 corresponding to spinal cord level L6–S1 in the rat). The cannula was secured in place with silicone elastomer (Kwik‐sil; World Precision Instruments, FL, USA) to create a tight seal at the point of cannulation. There was no cerebrospinal fluid leak. All of the drugs used in the experiments, except atropine, as well as the drugs used for anaesthesia, were intrathecally applied via the cannula positioned at the L6–S1 spinal cord level. For i.v. injection of atropine, the femoral vein was cannulated. Atropine injection was started 10 min before intrathecal dopamine administration.

Spinal cord slice preparation and patch clamp recordings

The method for obtaining spinal cord slices has been described previously (Yoshimura & Nishi, 1993). Briefly, rats (1–3 weeks old) were deeply anaesthetized with urethane (1.2–1.5 g kg−1, i.p. injection) and then thoracolumbar laminectomy was performed. The lumbosacral spinal cord was removed and placed in a pre‐oxygenated cold Krebs solution containing (in mm): 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3 and 11 glucose at 1–3°C. The pia‐arachnoid membrane was removed after cutting all of the ventral and dorsal roots. The spinal cord was mounted on a vibratome and then a 550 μm‐thick horizontal slice was cut. The slice was placed in the recording chamber and then perfused at a rate of 10 ml min−1 with Krebs solution saturated with 95% O2 and 5% CO2 at 36 ± 1°C. Drugs were dissolved in Krebs solution and applied by perfusing via a three‐way stopcock without any alteration in the flow rate or temperature.

For recording from preganglionic neurons in the sacral parasympathetic nuclei (SPN), rats were injected with the retrograde tracer fast DiI (FD) (1,1′‐dilinoleyl‐3,3,3′,3′‐tetramethylindocarbocyanine, 4‐chlorobenzenesulphonate; Molecular Probes, Eugene, OR, USA; 10 μl, 20 μg μl−1 solved in DMSO) into the peritoneal cavity near the colorectum and anus at days 7–10 after birth and then returned to their home cages. Three to 7 days after the tracer injection, a horizontal slice of the lumbosacral spinal cord was cut. FD‐labelled neurons were visualized in the SPN using an upright microscope (BX51WI; Olympus Optical, Tokyo, Japan) equipped with infrared differential interference contrast Nomarski with a fluorescence filter (U‐MWIGA3; Olympus). Whole‐cell patch clamp recordings were made from parasympathetic neurons with K‐gluconate pipette solution containing (in mm): 135 K‐gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 Hepes and 5 Mg‐ATP (pH 7.2). Signals were acquired with a patch clamp amplifier (Axopatch 200B; Molecular Devices, Union City, CA, USA). The data were digitized with an analogue‐to‐digital converter (digidata 1321A; Molecular Devices). The firing properties of the neurons in response to current injections from the recording pipettes were examined under a current clamp condition. Membrane potentials and currents were recorded under current and voltage clamp conditions, respectively.

Double immunofluorescence

Rats (8 weeks old, n = 3) were anaesthetized with pentobarbital sodium (50 mg kg−1; i.p. injection) and perfused transcardially with Ringer solution (200 ml) followed by 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4, 200 ml). Spinal cords at L6–S1 were then removed and immersed in the same fixative for an additional 6–8 h at 4ºC. After washes in phosphate‐buffered saline (PBS) (pH 7.4), tissue samples were soaked in 30% sucrose in PBS for 8 h at 4ºC and frozen. The samples was coronally sectioned at a thickness of 20 μm with a cryostat and mounted on glass slides coated with chrome alum‐gelatin.

We performed double‐staining for dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) to clarify the localization of D2R in the rat spinal cord. Sections were rinsed in PBS (3 × 5 min) and incubated for 30 min at room temperature with non‐immune donkey serum (dilution 1:50). After incubation, sections were washed in PBS (3 × 5 min), incubated for 12 h with a mixture of rabbit antibody against D2R (dilution 1:200, D2R‐Rb‐Af; Frontier Science, Sapporo, Japan) and goat antibody against ChAT (dilution 1:200, AB144P; Merck Millipore, Billerica, MA, USA), and then rinsed in PBS (3 × 5 min). The sections were then incubated for 2 h with Alexa488‐labelled donkey anti‐rabbit IgG (dilution 1:200, 711‐545‐152; Jackson Immunoresearch, West Grove, PA, USA) and Cy3‐labelled donkey anti‐goat IgG (dilution 1:100, 705‐165‐147; Jackson Immunoresearch) and washed in PBS (3 × 5 min). They were then counterstained with 4′,6‐diamidino‐2‐phenylindole (Dojindo, Kumamoto, Japan) and coverslipped with Fluoromount (Diagnostic BioSystems, Pleasanton, CA, USA).

The anti‐D2R antibody was raised in rabbit against a synthetic polypeptide of 270–370 amino acid residues of the mouse D2R (Narushima et al. 2006). The D2R antibodies have also been used in immunohistochemical analyses of brain tissue (Narushima et al. 2006; Uchigashima et al. 2007). The anti‐ChAT antibody was produced in goat against the enzyme present in the human placenta. Immunoblotting analyses of extracts from rat striatum with this antibody showed the presence of single band of 68 kDa (Pongrac & Rylett, 1998). This was used to identify lumbosacral parasympathetic neurons in the rat spinal cord (Vera & Nadelhaft, 2000; Sun et al. 2009).

The sections were observed using an epifluorescence microscope (80i; Nikon, Tokyo, Japan) or a confocal laser microscope (C2; Nikon). By confocal microscopy, z‐stacks of confocal images were obtained. Projection images were made from five to 10 series at 1 μm intervals using NIS‐elements (Nikon). All images were adjusted and arranged using Photoshop CS5 (Adobe Systems Inc., San Jose, CA, USA) in addition to NIS‐Elements.

Reagents

The compounds used were: α‐chloralose (Nacalai Tesque, Inc., Kyoto, Japan), ketamine hydrochloride (Daiichi Sankyo Co., Ltd, Tokyo, Japan), dopamine, SCH23390, quinpirole, SKF38393, atropine sulphate salt monohydrate and TTX (Sigma, St Louis, MO, USA), and haloperidol (Sumitomo Dainippon Pharma Co., Ltd, Osaka, Japan). All powder drugs except α‐chloralose were dissolved in distilled water. α‐chloralose was solubilized with 10% 2‐hydroxypropyl‐β‐cyclodextrin (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and then made up with 0.9% saline for infusion.

Statistical analysis

Data are expressed as the mean ± SD. Statistical analyses were performed using paired, two‐tailed Student's t tests. P < 0.05 was considered statistically significant. In the in vivo experiment, dopamine‐induced responses were quantified using data obtained for an initial period of 5 min after the beginning of their appearance. When responses did not appear, data calculation was started 5 min after drug administration. For counting the number of contractions, all pressure increases > 2 mmHg above baseline were included.

Results

Effects of intrathecal injection of dopamine on colorectal motility

In a resting state before dopamine application, brief and small rises in intraluminal pressure, representing changes in contractile activity of the colorectum, occurred spontaneously without accompanying fluid output from the anal cannula. When 0.9% saline was injected into the lumbosacral spinal cord as a control, little or no change in colorectal motility was observed (Fig. 1 A). Subsequent injection of dopamine at doses of 0.02, 0.1 and 0.5 μmol significantly increased the frequency of colorectal contractions (0.02 μmol, 5.0 ± 2.5 contractions/5 min, P = 0.099; 0.1 μmol, 7.6 ± 2.6 contractions/5 min, P = 0.049; 0.5 μmol, 12.4 ± 3.2 contractions/5 min, P = 0.013; n = 5) (Fig. 1 B) compared to their respective vehicle controls (2.6 ± 0.9, 2.6 ± 1.7, 3.6 ± 2.3 contractions/5 min; n = 5) (Fig. 1 B). Dopamine also increased the expelled fluid volume (0.02 μmol, 1.81 ± 1.41 ml/5 min, P = 0.048; 0.1 μmol, 3.13 ± 2.50 ml/5 min, P = 0.049; 0.5 μmol, 3.13 ± 0.17 ml/5 min, P < 0.001; n = 5) compared to their respective vehicle controls (0.08 ± 0.11, 0.01 ± 0.01, 0.01 ± 0.01 ml/5 min; n = 5) (Fig. 1 C). These results indicate that intrathecal application of dopamine triggers propulsive contractions in the colorectum. i.v. injection of the muscalinic ACh receptor antagonist atropine (1 mg kg−1 −1h bolus i.v. and 6 mg kg−1 h−1 maintenance infusion i.v.) inhibited almost all of the effect of dopamine (0.5 μmol, intrathecally) on colorectal motility (data not shown), suggesting that the final mediator to induce smooth muscle contraction is mainly ACh released from enteric neurons. In line with a previous study (Lahlou, 1998), intrathecal injection of dopamine slightly decreased the blood pressure at the highest dose (0.5 μmol) (Fig. 1 A).

Figure 1. Responses of the colorectum to intrathecal dopamine applied to the lumbosacral spinal cord .

Figure 1

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A, representative recording traces of intraluminal pressure change (upper), expelled liquid volume (middle) and blood pressure (lower) after intrathecal application of dopamine (0.5 μmol) are shown. Vehicle denotes the time of 0.9% saline (10 μl) injection as a control. Bar graphs summarize the (B) frequency of contractions (contractions/5 min) and (C) expelled liquid volume (ml/5 min) after saline injection and after dopamine (0.02, 0.1 and 0.5 μmol) injection. Each value represents the mean ± SD (n = 5). *Significantly different from the vehicle control (P < 0.05).

Effects of TTX on dopamine‐enhanced colorectal motility

To examine whether the action of dopamine is neurogenic, the neuronal blocker TTX was intrathecally applied prior to the injection of dopamine. As shown in previous studies (Naitou et al. 2015 a,b), TTX (0.15 nmol, intrathecally, at the L6–S1 spinal level) caused no apparent changes in spontaneous contractions of the colorectum but did reduce blood pressure (data not shown). Under conditions in which neurons of the lumbosacral spinal cord were inactivated by intrathecal administration of TTX, intrathecal dopamine (0.5 μmol) failed to increase the frequency of contractions (Fig. 2 A) or the expelled fluid volume (data not shown).

Figure 2. Effects of pretreatment with TTX or transection of neural pathways on the colokinetic effect of dopamine .

Figure 2

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A, representative recording trace of intraluminal pressure change before and after injection of dopamine (0.5 μmol) under conditions of TTX‐induced neural inactivation is shown. B, representative recording trace of intraluminal pressure change before and after injection of dopamine (0.5 μmol) under conditions of neural disconnection between the brain stem and the spinal defaecation centre by transection of the thoracic spinal cord (T8 level) is shown. C, representative recording trace of intraluminal pressure change in response to the intrathecal injection of dopamine (0.5 μmol) after bilateral severing of the pelvic nerve is shown. Similar results were reproducibly obtained in three independent rats in each experiment.

Effects of surgical transection of the thoracic spinal cord at T8 on the colokinetic effect of dopamine

It is possible that supraspinal regions are involved in the effect of intrathecally injected dopamine because intracerebroventricularly injected dopamine receptor agonists increase colorectal contractions (Bueno et al. 1992). To examine whether the presence of supraspinal regions is essential for the action of intrathecally injected dopamine, the thoracic spinal cord was transected at the T8 level. One hour after transection of the thoracic spinal cord, 0.5 μmol of dopamine was injected into the L6–S1 spinal cord. As shown in Fig. 2 B, dopamine caused a marked propulsive motility of the colorectum.

Effects of surgical nerve transections on the dopamine‐induced enhancement of colorectal motility

To identify the nerve pathway that transmits the action of dopamine from the spinal defaecation centre to the colorectum, we performed bilateral transection of the pelvic nerve, which is derived from sacral parasympathetic preganglionic neurons. When the pelvic nerves were bilaterally transected, intrathecal dopamine (0.5 μmol) failed to enhance colorectal motility (Fig. 2 C).

Pharmacological characterization of dopamine receptor subtypes that mediate the action of dopamine in the spinal defaecation centre

We used D1‐like or D2‐like receptor‐selective agonists and antagonists to determine the receptor subtypes that are responsible for dopamine‐induced enhancement of colorectal motility in the spinal defaecation centre. Because the antagonists used in the present study work in a competitive manner, we used a lower dose of dopamine (0.3 μmol) in this experiment. Prior injection of the D2‐like receptor antagonist haloperidol (0.1 μmol, intrathecally) (Fig. 3 B), but not the D1‐like receptor antagonist SCH23390 (0.1 μmol, intrathecally) (Fig. 3 A), completely blocked the effect of subsequently injected dopamine (0.3 μmol, intrathecally). In agreement with this, the D2‐like receptor agonist quinpirole (0.3 μmol) injected in the spinal defaecation centre mimicked the action of dopamine (Fig. 3 D). By contrast, the D1‐like receptor agonist SKF38393 (0.3 μmol, intrathecally) exerted no effect (Fig. 3 C). These results demonstrate that spinal dopamine enhances colorectal motility via activation of D2‐like receptors.

Figure 3. Pharmacological investigation of the receptor family responsible for the effect of dopamine .

Figure 3

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Representative recording traces of intraluminal pressure change in response to the intrathecal injection of dopamine (0.3 μmol) with D1‐like or D2‐like dopamine receptor antagonist SCH23390 (0.1 μmol) or haloperidol (0.1 μmol) pretreatment are shown in (A) and (B), respectively. Representative recording traces of intraluminal pressure change before and after intrathecal application of the D1‐like or D2‐like dopamine receptor agonist SKF38993 (0.3 μmol) or quinpirole (0.3 μmol) are shown in (C) and (D), respectively. Similar results were reproducibly obtained in three independent rats in each experiment.

Actions of dopamine on spinal neurons in the sacral parasympathetic nuclei

A retrograde tracing method was used to identify preganglionic neurons of the SPN projecting to the colorectum. Three to 7 days after injection of FD at the colorectum and anus, the dye was detected in the dorsal horn surface and IML of the lumbosacral spinal cord, in agreement with a previous study (Vizzard et al. 2000). To determine how dopamine acts on preganglionic neurons in the SPN, we next examined dopamine‐induced postsynaptic action on the SPN neurons retrogradely labelled with FD (Fig. 4 A). In current clamp mode, the FD‐labelled neurons (n = 21) tested showed tonic (a regular firing evoked throughout the current pulse) or phasic (a delayed firing with a slow ramp depolarization) firings in response to current injections from the recording pipette (Fig. 4 B), as reported in a previous study (Miura et al. 2000). As shown in Fig. 4 C, dopamine application (100 μm) induced an inward current in eight of 13 FD‐labelled neurons in the presence of TTX (0.5 μm) in voltage clamp mode at a holding potential of −50 mV. In the remaining FD‐labelled neurons, dopamine did not elicit any detectable postsynaptic currents. A dopamine‐induced inward current was detected in both phasic and tonic firing FD‐labelled neurons (Fig. 4 C). We further examined whether dopamine excites FD‐labelled neurons. In the FD‐labelled neurons in which an inward current was induced in the voltage clamp mode, a lower concentration of dopamine (10 μm) (Fig. 4 D, left), as well as a lower concentration of quinpirole (2 μm) (Fig. 4 D, right), increased action potentials in current clamp mode. The frequency of action potential was significantly increased by dopamine (Fig. 4 D). In the same FD‐labelled neurons in which dopamine had such excitatory actions, the D2‐like receptor agonist quinpirole (2 μm), but not the D1‐like receptor agonist SKF38993 (2 μm), also induced an inward current or action potentials (n = 3) (Fig. 4 E). Because recorded neurons were exposed to drugs repetitively, we used a lower concentration of dopamine to reduce the influence of desensitization.

Figure 4. Excitatory action of dopamine on retrogradely labelled neurons of sacral parasympathetic nuclei .

Figure 4

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A, example showing a fluorescence‐labelled neuron identified with FD injection (upper) and the same neuron observed with differential interference contrast optics (lower photograph). B, examples of the firing properties of FD‐labelled SPN neurons. FD‐labelled neurons showed a tonic or phasic firing property in response to 1 s current pulses (40 pA incremental current steps from −70 pA to +170 pA) through the recording electrode from a membrane potential of −80 mV. C, representative dopamine‐induced inward current in FD‐labelled SPN neurons in the presence of TTX (left trace). A dopamine‐induced inward current was detected in both phasic and tonic‐type FD‐labelled neurons (right). D, dopamine (upper left trace) and D2‐like agonist quinpirole (upper right trace) increased action potentials in FD‐labelled SPN neurons. The lower left trace shows action potentials under the action of dopamine in an expanded time course. Dopamine significantly increased the firing frequency of FD‐labelled neurons (paired t test, *P < 0.05, n = 3). E, example showing inward currents elicited by dopamine and D2‐like receptor agonist quinpirole, but not D1‐like receptor agonist SKF38993, in the same FD‐labelled neuron.

Immunohistochemistry

Punctate labelling of D2R immunoreactivity was ubiquitously observed in the spinal cord at L6–S1 (Fig. 5 A). In the SPN, D2R‐immunoreactive nerve cell bodies were densely distributed (Fig. 5 B). Some polygonal cells with D2R immunoreactivity were also immunoreactive to ChAT, although others were not (Fig. 5 C–E). The ratio of D2R‐immunoreactive cells in ChAT‐immunoreactive cells was 89.7% (126 cells having been counted). Punctate labelling was also observed in the neuropil of the SPN.

Figure 5. Localization of D2‐like dopamine receptors in the sacral parasympathetic nuclei .

Figure 5

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Double immunofluorescence images of D2R (green) and ChAT (red). A, lower magnification view of the spinal cord at L6–S1. Immunoreactivity for D2R was observed all over the spinal cord at the L6–S1 level. ChAT‐immnoreactivity was observed in the SPN (A, arrow), ventral horn and around the central canal. B, Double immunofluorescence images of D2R and ChAT in the SPN. There are D2R‐immunoreactive neurons in the SPN and some of them are also ChAT‐immunoreactive. C–E, higher magnification view of the SPN marked in (B). Immunoreactivity for D2R (C), ChAT (D) and merged (E). Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (blue). Scale bars = 200 μm in (A); 20 μm in (B); 10 μm in (C) to (D).

Discussion

In the present study, we have demonstrated, for the first time to our knowledge, that intrathecal application of dopamine to the L6–S1 region of the spinal cord enhanced colorectal motility. The major findings regarding the mechanism of action are: (1) the effect of dopamine was abolished by TTX‐induced inactivation of spinal defaecation centre or by bilateral severing of the pelvic nerves but not by transection of the T8 thoracic spinal cord; (2) the dopamine D2‐like receptor agonist mimicked, and its antagonist completely blocked, the effect of dopamine; (3) dopamine activated preganglionic neurons of SPN via D2‐like receptors in the presence of TTX; and (4) the D2‐like receptors are expressed in the SPN neurons. These findings show that dopamine D2‐like receptors on preganglionic neurons of SPN mediated the action of intrathecal dopamine.

It appears that dopamine acts on neurons of the spinal defaecation centre because the effect of dopamine disappeared after inactivation of spinal neurons by local injection of TTX at the L6–S1 level (Fig. 2 A). This is further supported by the finding that dopamine induces neuronal responses in slice preparations of the lumbosacral spinal cord (Fig. 4). It should be noted, however, that these findings do not necessarily rule out the possible involvement of supraspinal regions in the action of dopamine. Indeed, dopamine has an influence on the sensory process in the spinal cord (Millan, 2002). Thus, it is possible that the neurons activated in response to the intrathecal injection of dopamine are neurons of ascending pathways from the spinal cord to the brain, and transmitting signals to the supraspinal regions are essential to exert prokinetic action. However, this is not the case because intrathecally injected dopamine at the L6–S1 level caused propulsive motility of the colorectum even after spinal transection at the T8 thoracic cord (Fig. 2 B). Accordingly, it is most probable that intrathecally injected dopamine directly activates outflow pathways at the spinal defaecation centre without any relation to the brain.

Surgical cutting of the pelvic nerve abolished the colokinetic action of intrathecal dopamine (Fig. 2 C), suggesting that the action is mediated by the pelvic nerve, which is the sacral parasympathetic nerve. Pharmacological experiments revealed that the receptor subtype responsible for mediating the dopamine‐evoked enhancement of colorectal motility is the D2‐like receptor (Fig. 3). D2R, the major subtype of D2‐like receptors, is expressed in the IML of the lumbosacral spinal cord (Fig. 5), in which preganglionic neurons of pelvic nerves exist. Furthermore, dopamine, as well as the D2‐like receptor agonist quinpirole, evoked an inward current in FD‐labelled neurons of the SPN in which preganglionic neurons innervating the colorectum existed (Fig. 4). Because TTX failed to block the inward current evoked by dopamine, dopamine probably directly activates the neurons via D2‐like receptors. This is apparently inconsistent with the general concept that D2‐like receptors suppress the activity of neurons (Missale et al. 1998; Vallone et al. 2000). However, there is evidence to show that D2‐like dopamine receptors can activate a non‐selective cation channel (Haj‐Dahmane, 2001; Aman et al. 2007). Taken together, it is reasonable to assume that intrathecally injected dopamine directly activates preganglionic neurons of the sacral parasympathetic nerves to exert its colokinetic action.

Immunohistochemical findings showed that some polygonal cells with D2R immunoreactivity were not immunoreactive to ChAT (Fig. 5). It has been reported that D2‐like dopamine receptors are expressed in inhibitory GABAergic interneurons in the lumbar spinal cord and mediate the dopamine‐induced anti‐nociceptive effect (Yang et al. 1996). Therefore, it is conceivable that the ChAT‐negative cells with D2R immunoreactivity are inhibitory interneurons. In accordance with this, in our slice patch clamp experiments, outward current was recorded in neurons that were not labelled with retrograde dye injected into the colorectum (data not shown). This is in agreement with observation by Greif et al. (1995) that D2 receptors potentiate K+‐currents in rat striatal neurons, and the responses would be related to the anti‐nociceptive effect of dopamine. However, it is also possible that suppression of the interneurons via D2‐like receptors plays a role in the colokinetic action of dopamine. For example, sacral parasympathetic outflow can be enhanced by suppressing inhibitory interneurons (i.e. producing disinhibition) in the spinal defaecation centre. This possibility remains to be clarified in future experiments.

Lumbar and thoracic spinal segments are virtually devoid of dopaminergic cell bodies (Millan, 2002). Although there are a few dopaminergic cell bodies in the cervical level and dorsal root ganglia in the rat, it is assumed that the dopaminergic innervation of the spinal cord is mainly derived from the brain (Millan, 2002). Karasawa et al. (2014) reported that disruption of brain dopaminergic neurons using microinjection of 6‐hydroxydopamine into the median forebrain bundle decreased faecal output and lead to constipation. Moreover, c‐Fos expression in the IML of the lumbosacral spinal cord was also decreased by denervation of brain dopaminergic neurons (Karasawa et al. 2014). These studies are consistent with our findings and allowed us to conclude that central dopamine systems are essential for normal defaecation. It has been shown that constipation occurs with 60–80% prevalence rates in PD patients (Jost, 2010; Sakakibara et al. 2011). Dysfunction of the ENS and/or vagal control is considered to be associated with constipation in PD patients. Our results may provide an additional aetiological relationship between degradation of dopaminergic neurons in the CNS and constipation. Degeneration of dopaminergic neurons brings about a reduction of dopamine in the spinal defaecation centre, and this subsequently leads to insufficient rectal contractions, resulting in constipation. Of particular importance is that bowel dysfunction affects the quality of life in patients with PD (Sakakibara et al. 2001). It has been reported that severe constipation in patients with PD is often resistant to present therapy including l‐DOPA administration (Jost, 2010). Considering that the therapeutic efficacy of l‐DOPA is probably a result of the release of dopamine by serotonergic neurons in which l‐DOPA is converted into dopamine (Arai et al. 1995; LeWitt, 2015), it can be speculated that compensatory serotonergic axon terminals do not exist in the spinal defaecation centre. Therefore, we expect that direct activation of the dopaminergic receptors in the spinal defaecation centre might be a novel target of pharmacological therapy for constipation in PD patients.

In our previous study, we demonstrated that intrathecally injected noradrenaline caused propulsive contractions of the colorectum via activation of the lumbosacral spinal defaecation centre, leading to a novel hypothesis that descending pain inhibitory pathways control not only pain, but also the defaecation reflex in the lumbosacral spinal cord (Naitou et al. 2015 b). In the present study, we investigated the effects of the intrathecal injection of dopamine on colorectal motility to confirm our hypothesis. The results obtained clearly show that dopamine acting on neurons in the spinal defaecation centre through the D2‐like dopamine receptors activates colorectal motility. Dopamine is also known to be a neurotransmitter of the descending pain inhibitory pathways, and dopaminergic innervation of the spinal cord is mainly derived from the brain (Millan, 2002). Moreover, both acute and sustained noxious inputs accelerate dopamine release in the dorsal horn of the lumbar spinal cord (Weil‐Fugazza & Godefroy, 1993; Gao et al. 2001). Based on these results, it is rational to conclude that, in addition to descending noradrenergic pathways, descending dopaminergic pathways affect the defaecation reflex in the lumbosacral defaecation centre. Because the descending noradrenergic neurons and dopaminergic neurons arise from different parts of the brain, it is natural that these components of descending pain inhibitory pathways operate in response to different stimuli (Millan, 2002). Thus, we consider that dopamine and noradrenaline would exert their prokinetic actions individually or simultaneously depending on the stimuli (e.g. thermal, chemical or mechanical stimuli) and/or timing (e.g. acute or chronic). Further experiments are needed to reveal the specific roles of dopamine and noradrenaline.

In summary, we have shown that dopamine acting on neurons at the lumbosacral defaecation centre causes propulsive motility of the colorectum in rats. Because dopamine is a neurotransmitter of the descending pain inhibitory pathways, our findings provide evidence for the involvement of the pathways in the regulation of colorectal motility. The dopaminergic system in the spinal defaecation centre may be a valuable therapeutic target because excess operation of the descending dopaminergic pathways in response to chronic abdominal pain may be related to a defaecation disorder in irritable bowel syndrome patients and insufficiency of the pathway as a result of the degradation of central dopaminergic neurons in PD patients would be a cause of constipation.

Additional information

Competing interests

The authors declare that they have no competing interests.

Author contributions

KN, HN, AI, YN, YSa, TY, AY and NA contributed to collection, analysis and interpretation of data and wrote the article. TS, YY and HF participated in the design of the study and helped to draft the manuscript. YSh was a supervisor in this study and revised the article critically for important intellectual content. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This research was supported in part by Grants‐in‐Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (22380157 and 26292164).

Linked articles This article is highlighted by a Perspective by Sanger. To read this Perspective, visit http://dx.doi.org/10.1113/JP272560.

This is an Editor's Choice article from the 1 August 2016 issue.

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