Optimization of an autaptic culture system for studying cholinergic synapses in sympathetic ganglia.

An autaptic synapse (or 'autapse') is a functional connection between a neuron and itself, commonly used in studying the molecular mechanisms underlying synaptic transmission and plasticity in central neurons. Most previous studies on autonomic synaptic functions have relied on spontaneous connections among neurons in mass cultures. However, growing evidence supports the utility of microcultures cultivating autaptic neurons for examining cholinergic transmission within sympathetic ganglia. Despite these advancements, standardized protocols for culturing autaptic sympathetic neurons have yet to be established. Drawing on historical literature, this study delineates optimal experimental conditions to efficiently and reliably produce cholinergic synapses in sympathetic neurons within a short time frame. Our research emphasizes five key factors: (i) the generation of uniformly sized microislands of growth permissive substrates; (ii) the addition of nerve growth factor, ciliary neurotrophic factor (CNTF), and serum to the culture medium; (iii) independence from specific serum and neuronal medium types; (iv) the reciprocal roles of CNTF and glial cells; and (v) the promotion of cholinergic synaptogenesis in SCG neurons through indirect glia co-cultures, rather than direct glial feeder layer cultures. In conclusion, glia-free monocultures of SCG neurons are relatively simple to prepare and yield robust and reliable synaptic currents. This makes them an effective model system for straightforwardly addressing fundamental questions about neurogenic mechanisms involved in cholinergic synaptic transmission in autonomic ganglia. Furthermore, autaptic culture experiments could eventually be implemented to investigate the roles of functional neuron-satellite glia units in regulating cholinergic functions under physiological and pathological conditions.

Store-operated calcium entry in the satellite glial cells of rat sympathetic ganglia.

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca2+ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca2+ entry (SOCE) which is critical for cellular Ca2+ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca2+ stores were depleted in the absence of extracellular Ca2+, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca2+ entry channels. Intracellular Ca2+ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca2+ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.

Increased expression of neuregulin 1 in the urothelium of rat bladder with partial bladder outlet obstruction.

BACKGROUND: This study determined whether changes in the expression of neuregulin (NRG) 1, erbB2 tyrosine kinase (ErbB2) and the M2 muscarinic receptor in the urothelium and detrusor muscle of the rat bladder were associated with partial bladder outlet obstruction (PBOO). METHODS: Male Sprague-Dawley rats (body weight 250-300 g) were used and subdivided into control (n = 10) and PBOO groups (n = 20). PBOO was induced for 21 days, and the expression of NRG1, ErbB2 and M2 muscarinic receptor mRNA and protein was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting, respectively. RESULTS: In the urothelium of rat bladder samples, protein expression and mRNA expression of NRG1, ErbB2 and M2 muscarinic receptor were significantly increased in the PBOO group compared to the control group (p < 0.05). Only mRNA expression levels of NRG1/ ErbB2 were higher in the detrusor muscle of the PBOO group compared to the control group (p < 0.05). CONCLUSIONS: Our study demonstrated remarkable changes in the expression of NRG1/ErbB2 receptor mRNA and protein in the urothelium and muscle layer. These results suggest that NRG1 overexpression plays some kind of role against the PBOO-induced upregulated muscarinic receptors in detrusor overactivity.

Decreased excitability and voltage-gated sodium currents in aortic baroreceptor neurons contribute to the impairment of arterial baroreflex in cirrhotic rats.

Cardiovascular autonomic dysfunction, which is manifested by an impairment of the arterial baroreflex, is prevalent irrespective of etiology and contributes to the increased morbidity and mortality in cirrhotic patients. However, the cellular mechanisms that underlie the cirrhosis-impaired arterial baroreflex remain unknown. In the present study, we examined whether the cirrhosis-impaired arterial baroreflex is attributable to the dysfunction of aortic baroreceptor (AB) neurons. Biliary and nonbiliary cirrhotic rats were generated via common bile duct ligation (CBDL) and intraperitoneal injections of thioacetamide (TAA), respectively. Histological and molecular biological examinations confirmed the development of fibrosis in the livers of both cirrhotic rat models. The heart rate changes during phenylephrine-induced baroreceptor activation indicated that baroreflex sensitivity was blunted in the CBDL and TAA rats. Under the current-clamp mode of the patch-clamp technique, cell excitability was recorded in DiI-labeled AB neurons. The number of action potential discharges in the A- and C-type AB neurons was significantly decreased because of the increased rheobase and threshold potential in the CBDL and TAA rats compared with sham-operated rats. Real-time PCR and Western blotting indicated that the NaV1.7, NaV1.8, and NaV1.9 transcripts and proteins were significantly downregulated in the nodose ganglion neurons from the CBDL and TAA rats compared with the sham-operated rats. Consistent with these molecular data, the tetrodotoxin-sensitive NaV currents and the tetrodotoxin-resistant NaV currents were significantly decreased in A- and C-type AB neurons, respectively, from the CBDL and TAA rats compared with the sham-operated rats. Taken together, these findings implicate a key cellular mechanism in the cirrhosis-impaired arterial baroreflex.

Neuregulin 1 as an endogenous regulator of nicotinic acetylcholine receptors in adult major pelvic ganglion neurons.

We investigated whether endogenous neuregulin 1 (NRG1) is released in a soluble form (called sNRG1) and upregulates expression of nicotinic acetylcholine receptor (nAChR) in autonomic major pelvic ganglion (MPG) neurons of adult rats. To elicit the release of sNRG1, either the hypogastric nerve or the pelvic nerve was electrically stimulated. Then, the MPG-conditioned medium (CM) was subjected to western blotting using an antibody directed against the N-terminal ectodomain of NRG1. Both sympathetic and parasympathetic nerve activation elicited the release of sNRG1 from MPG neurons in a frequency-dependent manner. The sNRG1 release was also induced by treatment of MPG neurons with either high KCl or neurotrophic factors. The biological activity of the released sNRG1 was detected by tyrosine phosphorylation (p185) of the ErbB2 receptors in MPG neurons. When MPG neurons were incubated for 6 h in the CM, the protein level of the nAChR α3 subunit and ACh-induced current (IACh) density were significantly increased. The CM-induced changes in IACh was abolished by a selective ErbB2 tyrosine kinase inhibitor. Taken together, these data suggest that NRG1 functions as an endogenous regulator of nAChR expression in adult MPG neurons.

Cellular and Molecular Mechanisms Underlying Altered Excitability of Cardiac Efferent Neurons in Cirrhotic Rats.

Patients with cirrhosis often exhibit cardiac autonomic dysfunction (CAD), characterized by enhanced cardiac sympathetic activity and diminished cardiac vagal tone, leading to increased morbidity and mortality. This study delineates the cellular and molecular mechanisms associated with altered neuronal activities causing cirrhosis-induced CAD. Biliary and nonbiliary cirrhotic rats were produced by common bile duct ligation (CBDL) and intraperitoneal injections of thioacetamide (TAA), respectively. Three weeks after CBDL or TAA injection, the assessment of heart rate variability revealed autonomic imbalance in cirrhotic rats. We observed increased excitability in stellate ganglion (SG) neurons and decreased excitability in intracardiac ganglion (ICG) neurons in cirrhotic rats compared to sham-operated controls. Additionally, threshold, rheobase, and action potential duration exhibited opposite alterations in SG and ICG neurons, along with changes in afterhyperpolarization duration. A- and M-type K⁺ channels were significantly downregulated in SG neurons, while M-type K⁺ channels were upregulated, with downregulation of the N- and L-type Ca2⁺ channels in the ICG neurons of cirrhotic rats, both in transcript expression and functional activity. Collectively, these findings suggest that cirrhosis induces an imbalance between cardiac sympathetic and parasympathetic neuronal activities via the differential regulation of K+ and Ca2+ channels. Thus, cirrhosis-induced CAD may be associated with impaired autonomic efferent functions within the homeostatic reflex arc that regulates cardiac functions.

Changes in the expression of satellite glial cell-specific markers during postnatal development of rat sympathetic ganglia.

The sympathetic ganglia represent a final motor pathway that mediates homeostatic "fight and flight" responses in the visceral organs. Satellite glial cells (SGCs) form a thin envelope close to the neuronal cell body and synapses in the sympathetic ganglia. This unique morphological feature suggests that neurons and SGCs form functional units for regulation of sympathetic output. In the present study, we addressed whether SGC-specific markers undergo age-dependent changes in the postnatal development of rat sympathetic ganglia. We found that fatty acid-binding protein 7 (FABP7) is an early SGC marker, whereas the S100B calcium-binding protein, inwardly rectifying potassium channel, Kir4.1 and small conductance calcium-activated potassium channel, SK3 are late SGC markers in the postnatal development of sympathetic ganglia. Unlike in sensory ganglia, FABP7 + SGC was barely detectable in adult sympathetic ganglia. The expression of connexin 43, a gap junction channel gradually increased with age, although it was detected in both SGCs and neurons in sympathetic ganglia. Glutamine synthetase was expressed in sensory, but not sympathetic SGCs. Unexpectedly, the sympathetic SGCs expressed a water-selective channel, aquaporin 1 instead of aquaporin 4, a pan-glial marker. However, aquaporin 1 was not detected in the SGCs encircling large neurons. Nerve injury and inflammation induced the upregulation of glial fibrillary acidic protein, suggesting that this protein is a hall marker of glial activation in the sympathetic ganglia. In conclusion, our findings provide basic information on the in vivo profiles of specific markers for identifying sympathetic SGCs at different stages of postnatal development in both healthy and diseased states.

CRISPR/Cas9-mediated knock-in of a fluorescent reporter into the target locus of interest in human pluripotent stem cells.

The method presented herein is associated with the Lab Resource article titled "Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3, using CRISPR/CAS9-based gene targeting" [1]. The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is encoded by the human MYH6 gene, which is expressed in both the atria and ventricles during embryonic development and is predominantly expressed in the atria after birth [2]. Herein, the methods used to achieve CRISPR/SpCas9-mediated introduction of an EGFP reporter into αMHC, the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described. The CRISPR-Cas9 system enables efficient replacement of the stop codon in the last exon of αMHC with a 2A non-joining peptide (T2A)-EGFP cassette. First, hPSCs are transfected with the donor construct and Cas9/sgRNA plasmids via electroporation and selected with neomycin for approximately 3 weeks. Thereafter, the established cell line exhibits typical characteristics of human embryonic stem cells (hESCs). When these cells differentiate into cardiomyocytes, the expression of EGFP is confirmed using confocal microscopy, flow cytometry analysis, and immunostaining.•The line enables monitoring of cell maturation events during human cardiac development.•The line is a valuable platform for cardiotoxicity tests and drug screening.•This method has already been employed in two original studies, as previously reported for reporter cell line generation using CRISPR/Cas9.

Neuregulin 1 up-regulates the expression of nicotinic acetylcholine receptors through the ErbB2/ErbB3-PI3K-MAPK signaling cascade in adult autonomic ganglion neurons.

We investigated effects of Neuregulin 1 (NRG1) on the expression of nicotinic acetylcholine receptor (nAChR) in major pelvic ganglion (MPG) from adult rat. MPG neurons were found to express transcripts for type I and III NRG1s as well as α and ß-type epidermal growth factor (EGF)-like domains. Of the four ErbB receptor isoforms, ErbB1, ErbB2, and ErbB3 were expressed in MPG neurons. Treating MPG with NRG1ß significantly increased the transcript and protein level of the nAChR α3 and ß4 subunits. Consistent with these molecular data, nicotinic currents (I(ACh) ) were significantly up-regulated in NRG1ß-treated sympathetic and parasympathetic MPG neurons. In contrast, the type III NRG1 and the α form of the NRG1 failed to alter the I(ACh) . Inhibition of the ErbB2 tyrosine kinase completely abolished the effects of NRG1ß on the I(ACh) . Stimulation of the ErbB receptors by NRG1ß activated the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK). Immunoblot analysis revealed that PI3K-mediated activation of Akt preceded Erk1/2 activation in NRG1ß-treated MPG neurons. Furthermore, specific PI3K inhibitors abrogated the phosphorylation of Erk1/2, while inhibition of MEK did not prevent the phosphorylation of Akt. Taken together, these findings suggest that NRG1 up-regulates nAChR expression via the ErbB2/ErbB3-PI3K-MAPK signaling cascade and may be involved in maintaining the ACh-mediated synaptic transmission in adult autonomic ganglia.

Functional plasticity of cardiac efferent neurons contributes to traumatic brain injury-induced cardiac autonomic dysfunction.

Traumatic brain injury (TBI) frequently causes cardiac autonomic dysfunction (CAD), irrespective of its severity, which is associated with an increased morbidity and mortality in patients. Despite the significance of probing the cellular mechanism underlying TBI-induced CAD, animal studies on this mechanism are lacking. In the current study, we tested whether TBI-induced CAD is associated with functional plasticity in cardiac efferent neurons. In this regard, TBI was induced by a controlled cortical impact in rats. Assessment of heart rate variability and baroreflex sensitivity indicated that CAD was developed in the sub-acute period after moderate and severe TBI. The cell excitability was increased in the stellate ganglion (SG) neurons and decreased in the intracardiac ganglion (ICG) neurons in TBI rats, compared with the sham-operated rats. The transient A-type K+ (KA) currents, but not the delayed rectifying K+ currents were significantly decreased in SG neurons in TBI rats, compared with sham-operated rats. Consistent with these electrophysiological data, the transcripts encoding the Kv4 α subunits were significantly downregulated in SG neurons in TBI rats, compared with sham-operated rats. TBI causes downregulation and upregulation of M-type K+ (KM) currents and the KCNQ2 mRNA transcripts, which may contribute to the hyperexcitability of the SG neurons and the hypoexcitability of the ICG neurons, respectively. In conclusion, the key cellular mechanism underlying the TBI-induced CAD may be the functional plasticity of the cardiac efferent neurons, which is caused by the regulation of the KA and/or KM currents.

Expression of GABAA receptor beta2/3 subunits in the rat major pelvic ganglion.

Several pharmacological and physiological studies have suggested that GABA(A) receptors (GABA(A) Rs) may exist in the rat major pelvic ganglion (MPG), a large coalescent pelvic ganglion that contains both sympathetic and parasympathetic components which innervates pelvic organs. However, the presence of GABA(A) R in the MPG has never been demonstrated directly by morphological studies. In the present study, we used immunohistochemistry to demonstrate the existence of GABA(A) R beta2/3 subunits for the first time in the rat MPG. We also analyzed the neurochemical properties of MPG neurons expressing GABA(A) R beta2/3 subunits. GABA(A) R beta2/3-immunoreactive (-IR) neurons occupied 27.4+/-7.0% of the whole neuronal population, and many of these (77.6%) were co-localized with tyrosine hydroxylase (TH). Likewise, most (86.5%) of TH-IR neurons were GABA(A) R beta2/3-positive. GABA(A) R beta2/3 subunits were also expressed in a few VIP- or NOS-IR neurons, the cholinergic or non-adrenergic, non-cholinergic (NANC) neurons. These results suggest that GABA(A) Rs are involved in the modulation of most sympathetic, noradrenergic neurons and also a subset of VIP and NOS neurons of the rat MPG.

Bladder outlet obstruction causes up-regulation of nicotinic acetylcholine receptors in bladder-projecting pelvic ganglion neurons.

Pelvic ganglion (PG) neurons relay sympathetic and parasympathetic signals to the lower urinary tract, comprising the urinary bladder and bladder outlet, and are thus essential for both storage and voiding reflexes. Autonomic transmission is mediated by activation of the nicotinic acetylcholine receptor (nAChR) in PG neurons. Previously, bladder outlet obstruction (BOO), secondary to benign prostatic hyperplasia, was found to increase soma sizes of bladder-projecting PG neurons. To date, however, it remains unknown whether these morphological changes are accompanied by functional plasticity in PG neurons. In the present study, we investigated whether BOO alters acetylcholine receptor (nAChR) transcript expression and current density in bladder PG neurons. Partial ligation of the rat urethra for six weeks induced detrusor overactivity (DO), as observed during cystometrical measurement. In rats exhibiting DO, membrane capacitance of parasympathetic bladder PG neurons was selectively increased. Real-time PCR analysis revealed that BOO enhanced the expression of the transcripts encoding the nAChR α3 and ß4 subunits in PG neurons. Notably, BOO significantly increased ACh-evoked current density in parasympathetic bladder PG neurons, whereas no changes were observed in sympathetic bladder and parasympathetic penile PG neurons. In addition, other ligand-gated ionic currents were immune to BOO in bladder PG neurons. Taken together, these data suggest that BOO causes upregulation of nAChR in parasympathetic bladder PG neurons, which in turn may potentiate ganglionic transmission and contribute to the development of DO.

Modulation of Ca(v)3.2 T-type Ca2+ channels by protein kinase C.

Although T-type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Ca(v)3.2 T-type channels reconstituted in Xenopus oocytes. Phorbol-12-myristate-13-acetate (PMA) strongly enhanced the amplitude of Ca(v)3.2 channel currents (approximately 3-fold). The augmentation effects were not mimicked by 4alpha-PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Ca(v)3.2 channel activity via activation of oocyte PKC.

Use of RGS-insensitive Galpha subunits to study endogenous RGS protein action on G-protein modulation of N-type calcium channels in sympathetic neurons.

Regulators of G-protein signaling (RGS) proteins are a large family of signaling proteins that control both the magnitude and temporal characteristics of heterotrimeric G-protein-mediated signaling. A current challenge is to define how endogenous RGS protein function impacts G-protein modulation of ionic channels in mammalian neurons. The experimental strategy described here utilizes distinct mutations in Galpha subunits that confer Bordetella pertussis toxin (PTX) and RGS protein insensitivity. The native signaling pathway in rat sympathetic neurons that mediates voltage-dependent modulation of N-type Ca2+ channels is ablated by PTX treatment and the signaling is reconstituted by expressing a PTX/RGS-insensitive Galpha mutant along with Gbeta and Ggamma subunits. As neurons are resistant to conventional transfection modalities, heterologous expression is accomplished by the direct microinjection of plasmids into the nucleus of the neuron. An advantage of this approach is that knowledge of the specific RGS subtypes participating in the pathway is not required. From the resulting alterations in the kinetics and pharmacology of G-protein-coupled receptor modulation of N-type Ca2+ channels, we can infer the role endogenous RGS proteins play in the signaling pathway.

Divalent metals differentially block cloned T-type calcium channels.

We tested divalent metals including Cu2+, Pb2+, and Zn2+ to determine their pharmacological profiles for blockade of cloned T-type Ca2+ channels (alpha1G, alpha1 H, and alpha1I). Effects of the metals were also evaluated for native low and high voltage-activated Ca2+ channels in rat sympathetic pelvic neurons. Cu2+ and Zn2+ blocked three T-type channel isoforms in a concentration-dependent manner with a higher affinity for alpha1H currents (IC50 = 0.9 microM and 2.3 microM). In pelvic neurons, only Zn2+ showed strong selectivity for T-type Ca2+ currents over high voltage-activated Ca2+ currents. Conversely, Pb2+ block on Ca2+ channels did not show distinctive selectivity. Taken together, these results suggest that besides Ni2+, Cu2+ and Zn2+ can be used as selective blockers of alpha1 H at low concentrations.

Nerve injury alters profile of receptor-mediated Ca2+ channel modulation in vagal afferent neurons of rat nodose ganglia.

Although nerve injury is known to up- and down-regulate some metabotropic receptors in vagal afferent neurons of the nodose ganglia (NG), the functional significance has not been elucidated. In the present study, thus, we examined whether nerve injury affected receptor-mediated Ca2+ channel modulation in the NG neurons. In this regard, unilateral vagotomy was performed using male Sprague-Dawley rats. One week after vagotomy, Ca2+ currents were recorded using the whole-cell variant of patch-clamp technique in enzymatically dissociated NG neurons. In sham controls, norepinephrine (NE)-induced Ca2+ current inhibition was negligible. Following vagotomy, however, the NE responses were dramatically increased. This phenomenon was in accordance with up-regulation of alpha2A/B-adrenergic receptor mRNAs as quantified using real-time RT-PCR analysis. In addition, neuropeptide Y (NPY) and prostaglandin E2 responses were moderately augmented in vagotomized NG neurons. The altered NPY response appears to be caused by up-regulation of Y2 receptors negatively coupled to Ca2+ channels. In contrast, nerve injury significantly suppressed opioid (tested with DAMGO)-induced Ca2+ current inhibition with down-regulation of micro-receptors. Taken together, these results demonstrated for the first time that the profile of neurotransmitter-induced Ca2+ channel modulation is significantly altered in the NG neurons under pathophysiological state of nerve injury.

Co-localization of activating transcription factor 3 and phosphorylated c-Jun in axotomized facial motoneurons.

Activating transcription factor 3 (ATF3) and c-Jun play key roles in either cell death or cell survival, depending on the cellular background. To evaluate the functional significance of ATF3/c-Jun in the peripheral nervous system, we examined neuronal cell death, activation of ATF3/c-Jun, and microglial responses in facial motor nuclei up to 24 weeks after an extracranial facial nerve axotomy in adult rats. Following the axotomy, neuronal survival rate was progressively but significantly reduced to 79.1% at 16 weeks post-lesion (wpl) and to 65.2% at 24 wpl. ATF3 and phosphorylated c-Jun (pc-Jun) were detected in the majority of ipsilateral facial motoneurons with normal size and morphology during the early stage of degeneration (1-2 wpl). Thereafter, the number of facial motoneurons decreased gradually, and both ATF3 and pc-Jun were identified in degenerating neurons only. ATF3 and pc-Jun were co-localized in most cases. Additionally, a large number of activated microglia, recognized by OX6 (rat MHC II marker) and ED1 (phagocytic marker), gathered in the ipsilateral facial motor nuclei. Importantly, numerous OX6- and ED1-positive, phagocytic microglia closely surrounded and ingested pc-Jun-positive, degenerating neurons. Taken together, our results indicate that long-lasting co-localization of ATF3 and pc-Jun in axotomized facial motoneurons may be related to degenerative cascades provoked by an extracranial facial nerve axotomy.

Phenotype-specific down-regulation of nicotinic acetylcholine receptors in the pelvic ganglia of castrated rats: implications for neurogenic erectile dysfunction.

Pelvic ganglia (PG) play critical roles in relaying sympathetic and parasympathetic information from the spinal cord to the penile vasculature and, controlling the penile reflex. Animal studies have shown that androgen deprivation by castration causes erectile dysfunction (ED). Until now, however, neural mechanisms underlying castration-induced ED remain unclear. Therefore, we examined whether androgen deprivation down-regulates nicotinic acetylcholine receptors (nAchRs), which mediate fast excitatory synaptic transmission in the PG. Toward this end, neurogenic ED was demonstrated by measuring the intracavernous pressure in castrated rats. Real-time PCR analysis revealed that the transcripts encoding nAchR α3/α5/ß4 subunits were significantly down-regulated in the PG neurons. In addition, down-regulation of the nAchR subunits was reversed by replacement of testosterone. Patch-clamp experiments showed that the nAchR currents were selectively attenuated in the parasympathetic PG neurons innervating the penile vasculature, activation of which elicits penile erection. Taken together, our data suggest that phenotype-specific down-regulation of nAchRs in the PG neurons may contribute to the neurogenic ED in castrated rats.

Switching-on of serotonergic calcium signaling in activated hepatic stellate cells.

AIM: To investigate serotonergic Ca²+ signaling and the expression of 5-hydroxytryptamine (5-HT) receptors, as well as Ca²+ transporting proteins, in hepatic stellate cells (HSCs). METHODS: The intracellular Ca²+ concentration [Ca²+](i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system. Quantitative PCR was performed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum (ER) proteins involved in Ca²+ storage and release in cultured rat HSCs. RESULTS: Distinct from quiescent cells, activated HSCs exhibited [Ca²+](i) transients following treatment with 5-HT, which was abolished by U-73122, a phospholipase C inhibitor. Upregulation of 5-HT(2A) and 5-HT(2B) receptors, but not 5-HT3, was prominent during trans-differentiation of HSCs. Pretreatment with ritanserin, a 5-HT2 antagonist, inhibited [Ca²+](i) changes upon application of 5-HT. Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca²+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca²+ storage and release in activated HSCs. Ca²+ binding chaperone proteins of the ER, including calreticulin, calnexin and calsequestrin, were up-regulated following activation of HSCs. CONCLUSION: The appearance of 5-HT-induced [Ca²+](i) response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca²+ transporting/chaperone ER proteins may participate in the activating process of HSCs.

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats.

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 microM to 1mM). ACh increased both intracellular Ca(2+) ([Ca(2+)](i)) and Na(+) concentrations ([Na(+)](i)) in MPG neurons. The recovery of [Na(+)](i) after the removal of ACh was markedly delayed by ouabain (100 microM), an inhibitor of Na(+)/K(+) ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2+/-5.4% (n=7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca(2+)](i) with BAPTA/AM (20 microM) or the blockade of small-conductance Ca(2+)-activated K(+) channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na(+)](i) and [Ca(2+)](i), which activates Na(+)/K(+) ATPase and Ca(2+)-activated K(+) channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.