Neurons In Sacral Parasympathetic Nucleus Research Articles

PD23-05 THERAPEUTIC EFFECTS OF KCC2 CHLORIDE ION TRANSPORTER ACTIVATION ON DETRUSOR OVERACTIVITY IN MICE WITH SPINAL CORD INJURY

You have accessJournal of UrologyCME1 Apr 2023PD23-05 THERAPEUTIC EFFECTS OF KCC2 CHLORIDE ION TRANSPORTER ACTIVATION ON DETRUSOR OVERACTIVITY IN MICE WITH SPINAL CORD INJURY Kyohei Watanabe, Masaru Ishibashi, Takahisa Suzuki, Atsushi Otsuka, Naoki Yoshimura, Hideaki Miyake, and Atsuo Fukuda Kyohei WatanabeKyohei Watanabe More articles by this author , Masaru IshibashiMasaru Ishibashi More articles by this author , Takahisa SuzukiTakahisa Suzuki More articles by this author , Atsushi OtsukaAtsushi Otsuka More articles by this author , Naoki YoshimuraNaoki Yoshimura More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , and Atsuo FukudaAtsuo Fukuda More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003296.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: GABA generally exerts inhibitory effects in the central nervous system, including the spinal cord. However, it may also act as an excitatory transmitter under pathological conditions, such as injury. This paradoxical signaling of GABA is dependent on the intracellular Cl- concentration in neurons. K+-Cl-co-transporter 2 (KCC2) maintains a low intracellular Cl- concentration by pumping K+ and Cl- ions out of the cell. We aimed to clarify whether the down regulation of KCC2 in the sacral parasympathetic nucleus (SPN) of the lumbosacral spinal cord, from which the efferent pathway innervating the bladder originates, causes cellular hyperexcitability and triggers detrusor overactivity (DO) in spinal cord injury (SCI). METHODS: SCI was produced by the Th8-9 spinal cord transection in female C57BL/6 mice. We randomly divided mice into four groups: a spinal cord intact (SI)-vehicle, SI-CLP290, SCI-vehicle, and SCI-CLP290 groups. At 4 weeks after SCI, CLP290, a KCC2 activator was administered, and cystometry was performed. Thereafter, neuronal activity with c-fos staining and KCC expression in the cholinergic preganglionic parasympathetic neurons in the SPN were examined using immunohistochemistry. Firing properties of neurons in the SPN region were evaluated by extracellular recordings in spinal cord slice preparations. RESULTS: DO evident as non-voiding contractions (NVC) was significantly reduced by CLP290 treatment in SCI mice (Figure 1A-D). The number of c-fos-positive cells and co-expression of c-fos in choline acetyltransferase (ChAT)-positive cells were decreased in the SPN region of SCI-CLP290 group vs. SCI-vehicle group. KCC2 immunoreactivity was present on the cell membrane of SPN neurons, and the normalized fluorescence intensity of KCC2 in ChAT-positive SPN neurons was decreased in SCI-vehicle group, vs. SI-vehicle group, but recovered in SCI-CLP290 group. Extracellular recordings showed that CLP290 suppressed the high frequency firing activity of SPN neurons in SCI mice (Figure 1E-F). CONCLUSIONS: This study is the first report to suggest that activation of KCC2 chloride ion transporter may be a therapeutic modality for the treatment of SCI-induced DO by targeting bladder efferent pathways. Source of Funding: This work supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and a HUSM Grant-in-Aid © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e672 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kyohei Watanabe More articles by this author Masaru Ishibashi More articles by this author Takahisa Suzuki More articles by this author Atsushi Otsuka More articles by this author Naoki Yoshimura More articles by this author Hideaki Miyake More articles by this author Atsuo Fukuda More articles by this author Expand All Advertisement PDF downloadLoading ...

Therapeutic effects of KCC2 chloride transporter activation on detrusor overactivity in mice with spinal cord injury.

This study aimed to clarify whether downregulation of K+-Cl- cotransporter 2 (KCC2) in the sacral parasympathetic nucleus (SPN) of the lumbosacral spinal cord, from which the efferent pathway innervating the bladder originates, causes cellular hyperexcitability and triggers detrusor overactivity (DO) in spinal cord injury (SCI). SCI was produced by Th8-9 spinal cord transection in female C57BL/6 mice. At 4 wk after SCI, CLP290, a KCC2 activator, was administered, and cystometry was performed. Thereafter, neuronal activity with c-fos staining and KCC2 expression in cholinergic preganglionic parasympathetic neurons in the SPN was examined using immunohistochemistry. Firing properties of neurons in the SPN region were evaluated by extracellular recordings in the spinal cord slice preparations. DO evident as nonvoiding contractions was significantly reduced by CLP290 treatment in SCI mice. The number of c-fos-positive cells and coexpression of c-fos in choline acetyltransferase-positive cells were decreased in the SPN region of the SCI CLP290-treated group versus the SCI vehicle-treated group. KCC2 immunoreactivity was present on the cell membrane of SPN neurons and normalized fluorescence intensity of KCC2 in choline acetyltransferase-positive SPN neurons was decreased in the SCI vehicle-treated group versus the spinal intact vehicle-treated group but recovered in the SCI CLP290-treated group. Extracellular recordings showed that CLP290 suppressed the high-frequency firing activity of SPN neurons in SCI mice. These results indicated that SCI-induced DO is associated with downregulation of KCC2 in preganglionic parasympathetic neurons and that activation of KCC2 transporters can reduce DO, increase KCC2 expression in preganglionic parasympathetic neurons, and decrease neuronal firing of SPN neurons in SCI mice.NEW & NOTEWORTHY This study is the first report to suggest that activation of the Cl- transporter K+-Cl- cotransporter 2 may be a therapeutic modality for the treatment of spinal cord injury-induced detrusor overactivity by targeting bladder efferent pathways.

Enhanced Contractive Tension and Upregulated Muscarinic Receptor 2/3 in Colorectum Contribute to Constipation in 6-Hydroxydopamine-Induced Parkinson's Disease Rats.

Constipation and defecatory dysfunctions are frequent symptoms in patients with Parkinson’s disease (PD). The pathology of Lewy bodies in colonic and rectal cholinergic neurons suggests that cholinergic pathways are involved in colorectal dysmotility in PD. However, the underlying mechanism is unclear. The aim of the present study is to examine the effect of central dopaminergic denervation in rats, induced by injection 6-hydroxydopamine into the bilateral substania nigra (6-OHDA rats), on colorectal contractive activity, content of acetylcholine (ACh), vasoactive intestinal peptide (VIP) and expression of neural nitric oxide synthase (nNOS) and muscarinic receptor (MR). Strain gauge force transducers combined with electrical field stimulation (EFS), gut transit time, immunohistochemistry, ELISA, western blot and ultraperformance liquid chromatography tandem mass spectrometry were used in this study. The 6-OHDA rats exhibited outlet obstruction constipation characterized by prolonged transit time, enhanced contractive tension and fecal retention in colorectum. Pretreatment with tetrodotoxin significantly increased the colorectal motility. EFS-induced cholinergic contractions were diminished in the colorectum. Bethanechol chloride promoted colorectal motility in a dose-dependent manner, and much stronger reactivity of bethanechol chloride was observed in 6-OHDA rats. The ACh, VIP and protein expression of nNOS was decreased, but M2R and M3R were notably upregulated in colorectal muscularis externa. Moreover, the number of cholinergic neurons was reduced in sacral parasympathetic nucleus (SPN) of 6-OHDA rats. In conclusion, central nigrostriatal dopaminergic denervation is associated with decreased cholinergic neurons in SPN, decreased ACh, VIP content, and nNOS expression and upregulated M2R and M3R in colorectum, resulting in colorectal dysmotility, which contributes to outlet obstruction constipation. The study provides new insights into the mechanism of constipation and potential therapeutic targets for constipation in PD patients.

Sex differences in c-Fos and EGR-1/Zif268 activity maps of rat sacral spinal cord following cystometry-induced micturition.

Storage and voiding of urine from the lower urinary tract (LUT) must be timed precisely to occur in appropriate behavioral contexts. A major part of the CNS circuit that coordinates this activity is found in the lumbosacral spinal cord. Immediate early gene (IEG) activity mapping has been widely used to investigate the lumbosacral LUT‐related circuit, but most reports focus on the effects of noxious stimulation in anesthetized female rats. Here we use c‐Fos and EGR‐1 (Zif268) activity mapping of lumbosacral spinal cord to investigate cystometry‐induced micturition in awake female and male rats. In females, after cystometry c‐Fos neurons in spinal cord segments L5–S2 were concentrated in the sacral parasympathetic nucleus (SPN), dorsal horn laminae II–IV, and dorsal commissural nucleus (SDCom). Comparisons of cystometry and control groups in male and female revealed sex differences. Activity mapping suggested dorsal horn laminae II–IV was activated in females but showed net inhibition in males. However, inhibition in male rats was not detected by EGR‐1 activity mapping, which showed low coexpression with c‐Fos. A class of catecholamine neurons in SPN and SDCom neurons were also more strongly activated by micturition in females. In both sexes, most c‐Fos neurons were identified as excitatory by their absence of Pax2 expression. In conclusion, IEG mapping in awake male and female rats has extended our understanding of the functional molecular anatomy of the LUT‐related circuit in spinal cord. Using this approach, we have identified sex differences that were not detected by previous studies in anesthetized rats.

<i>In vivo</i> patch–clamp analysis of visceral spinal sensory synaptic transmission and spinal control of lower urinary tract function

Coordinated movement of the bladder, urethra and external urethral sphincter in the lower urinary tract (LUT) is essential for the bladder filling and voiding. The activations of these organs are precisely controlled by the central nervous system including the spinal cord, through the peripheral sympathetic (hypogastric), parasympathetic (pelvic) and somatic (pudendal) nerves. Preganglionic neurons in sacral parasympathetic nucleus (SPN) of the spinal cord play an important role of in regulating different pelvic organ function including micturition and defecation. In this study, we developed an in vivo patch-clamp recording technique from spinal preganglionic neurons to investigate how the spinal cord controls the LUT function at the synaptic level. Slice patch-clamp recordings from SPN neurons were also used to analyze excitatory monosynaptic inputs from primary afferent fibers. Preganglionic neurons in SPN showed tonic or phasic firing properties in response to current pulse injections and SPN neurons received monosynaptic Aδ and C fiber-mediated excitatory synaptic inputs. Intravesical pressure was synchronously changed with neuronal bursting of sacral spinal pre­ganglionic neurons in vivo. The newly developed recording technique is useful for elucidating the detailed synaptic mechanism for spinal control of LUT function.

Spinal and brain circuits to motoneurons of the bulbospongiosus muscle: Retrograde transneuronal tracing with rabies virus

Retrograde transneuronal tracing with rabies virus from the left bulbospongiosus muscle (BS) was used to identify the neural circuits underlying its peripheral and central activation. Rats were killed at 2, 3, 4, and 5 days post-inoculation (p.i.). Rabies immunolabelling was combined with immunohistochemical detection of choline acetyltransferase and oxytocin. Virus uptake was restricted to ipsilateral BS motoneurons (2 days p.i.). The onset of transfer (3 days p.i.) visualized interneurons in the dorsal grey commissure (DGC), intermediate zone, and sacral parasympathetic nucleus (SPN), mainly in DGC at L5–S1, and revealed synaptic connections between BS and external urethral sphincter motoneurons. At 4 and 5 days p.i., higher-order interneurons were labelled in other spinal areas and segments. Supraspinal labelling initially involved only Barrington's nucleus, nucleus reticularis magnocellularis, and paragigantocellularis lateralis (4 days p.i.). Later, labelling extended to other populations traditionally associated with control of sexual activity and micturition (periaqueductal grey, paraventricular nucleus, medial preoptic area, prefrontal cortex), but also indicated the intervention of somatic descending motor pathways (vestibulospinal and reticulospinal neurons, “hindlimb” regions of sensorimotor cortex and red nucleus) and cerebellar nuclei in multisynaptic innervation of the labelled motoneurons. Dual color immunofluorescence disclosed multisynaptic links between these motoneurons and thoracolumbar medial sympathetic (choline acetyltransferase-immunoreactive) neurons. In contrast, preganglionic neurons in SPN and most oxytocinergic neurons in paraventricular hypothalamic nucleus remained unlabelled, suggesting that parasympathetic and somatic outflow to pelvic organs are probably controlled by separate interneuronal populations and that oxytocinergic spinal projections are more likely to influence sacral autonomic rather than somatic outflow. J. Comp. Neurol. 414:167–192, 1999. © 1999 Wiley-Liss, Inc.

Spinal and brain circuits to motoneurons of the bulbospongiosus muscle: Retrograde transneuronal tracing with rabies virus

Retrograde transneuronal tracing with rabies virus from the left bulbospongiosus muscle (BS) was used to identify the neural circuits underlying its peripheral and central activation. Rats were killed at 2, 3, 4, and 5 days post-inoculation (p.i.). Rabies immunolabelling was combined with immunohistochemical detection of choline acetyltransferase and oxytocin. Virus uptake was restricted to ipsilateral BS motoneurons (2 days p.i.). The onset of transfer (3 days p.i.) visualized interneurons in the dorsal grey commissure (DGC), intermediate zone, and sacral parasympathetic nucleus (SPN), mainly in DGC at L5-S1, and revealed synaptic connections between BS and external urethral sphincter motoneurons. At 4 and 5 days p.i., higher-order interneurons were labelled in other spinal areas and segments. Supraspinal labelling initially involved only Barrington's nucleus, nucleus reticularis magnocellularis, and paragigantocellularis lateralis (4 days p.i.). Later, labelling extended to other populations traditionally associated with control of sexual activity and micturition (periaqueductal grey, paraventricular nucleus, medial preoptic area, prefrontal cortex), but also indicated the intervention of somatic descending motor pathways (vestibulospinal and reticulospinal neurons, "hindlimb" regions of sensorimotor cortex and red nucleus) and cerebellar nuclei in multisynaptic innervation of the labelled motoneurons. Dual color immunofluorescence disclosed multisynaptic links between these motoneurons and thoracolumbar medial sympathetic (choline acetyltransferase-immunoreactive) neurons. In contrast, preganglionic neurons in SPN and most oxytocinergic neurons in paraventricular hypothalamic nucleus remained unlabelled, suggesting that parasympathetic and somatic outflow to pelvic organs are probably controlled by separate interneuronal populations and that oxytocinergic spinal projections are more likely to influence sacral autonomic rather than somatic outflow.

The effect of rhizotomy on NADPH diaphorase staining in the lumbar spinal cord of the rat

In the L6-S1 spinal segments of the rat NADPH diaphorase fiber-like staining extending along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus was eliminated 1–3 weeks following dorsal-ventral L6-S1 rhizotomy. However, other NADPH diaphorase staining in the dorsal horn was not affected by rhizotomy. Rhizotomy also doubled the number (5 versus 10 cells/section) of NADPH diaphorase positive neurons in the region of the sacral parasympathetic nucleus (SPN). These data indicate that NADPH diaphorase is present in primary afferent projections to the SPN and that NADPH diaphorase activity in SPN neurons can be enhanced by neuronal injury. The functional significance of the NADPH diaphorase histochemical stain remains to be determined; however, if NADPH diaphorase is nitric oxide synthase then this would suggest that NO has an important role in the neural pathways to the pelvic viscera.

Neurons in the sacral parasympathetic nucleus that project to the hypothalamus do not also project through the pelvic nerve—a double labeling study combining Fluoro-gold and cholera toxin B in the rat

We recently reported that neurons in the sacral parasympathetic nucleus (SPN) project directly to the hypothalamus. In the present study, we examined the possibility that individual neurons in SPN send both an axon into the hypothalamus. We used a new double-labeling technique in which two sensitive retrograde tracers (Fluoro-gold and cholera toxin subunit B immunocytochemically stained with rhodamine-labeled antibodies) were combined. The effectiveness of this combination for singly and doubly labeling neurons was established in experiments in which both tracers were injected into overlapping areas of the tongue or ventrobasal thalamus. These injections doubly labeled large numbers of neurons in the hypoglossal or dorsal column nuclei, respectively. In studies of the projections of neurons in the SPN, injection of one tracer into the hypothalamus and the other into the pelvic nerve and/or pelvic ganglion singly labeled many neurons (more than 3300 in the 7 examined cases). However, no SPN neurons were doubly labeled. These findings indicate that the SPN in the rat consists of at least two distinct groups of cells, parasympathetic preganglionic neurons and neurons that project to the hypothalamus.

The distribution and morphology of parasympathetic preganglionic neurons in the cat sacral spinal cord as revealed by horseradish peroxidase applied to the sacral ventral roots.

Parasympathetic preganglionic neurons in the sacral parasympathetic nucleus (SPN) of the cat were studied by applying horseradish peroxidase (HRP) to the sacral ventral roots. The results were compared to data from earlier experiments in which these same neurons were labelled by HRP applied to the pelvic nerve at a point much further from the spinal cord. The present experiments have shown. The total number of neurons in the SPN determined by ventral root labelling is equal to the number obtained by pelvic nerve labelling. This indicates that virtually all SPN neurons send their axons into the pelvic nerve. More extensive dendritic projections from SPN neurons were revealed than in the pelvic nerve experiments. In particular, a strong dendritic projection extended within the lateral marginal zone of the dorsal horn close to Lissauer's tract and horizontal dendrites projected well into the contralateral gray matter, some reaching the contralateral sacral parasympathetic nucleus. The neurons labelled via any one ventral root were all contained within a region equal in length to one spinal segment but shifted rostrally by a small amount. Thus, no evidence was obtained for a long intraspinal pathway in which axons of spinal cord neurons entered ventral roots many segments away from their somata.