Muscarinic Cholinergic Agonist Research Articles

Dietary constituent, decanoic acid suppresses the excitability of nociceptive trigeminal neuronal activity associated with hypoalgesia via muscarinic M2 receptor signaling.

BackgroundAlthough decanoic acid (DA) is thought to act as a muscarinic cholinergic agonist, effect of DA on nociceptive behavioral responses and the excitability of nociceptive neuronal activity under in vivo conditions remain to be determined. The aim of the present study, therefore, was to investigate whether in vivo acute administration of ointment containing DA affects the excitability of nociceptive trigeminal spinal nucleus caudalis (SpVc) neurons associated with hypoalgesia in naïve rats.ResultsAfter local application of DA, the threshold of escape from mechanical stimulation applied to the shaved orofacial skin was significantly higher than before DA application. Vehicle treatment (without DA) had no significant effect on the escape threshold from mechanical stimulation. Extracellular single unit recordings were made from SpVc wide-dynamic range (WDR) neurons in response to orofacial non-noxious and noxious mechanical stimuli of pentobarbital-anesthetized rats. The mean firing frequency of SpVc WDR neurons in response to noxious, but not non-noxious, mechanical stimuli was inhibited by local application of DA, and the maximum inhibition of discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 1–5 min. The DA-induced short-term inhibitory effects were reversed after approximately 10 min. Pretreatment intravenously with the muscarinic-specific M2 receptor antagonist, methoctramine, abolished the DA-induced suppression of firing frequency of SpVc WDR neurons in response to noxious stimulation. Fluorogold (FG) labeling was identified as the trigeminal ganglion (TG) neurons innervating orofacial skin. FG-labeled small-diameter TG neurons expressed M2 receptor immunoreactivity.ConclusionThese results suggest that acute DA application induces short-term mechanical hypoalgesia and this effect was mainly due to suppression of the excitability of SpVc WDR neurons via the peripheral M2 receptor signaling pathway in the trigeminal primary afferents. These findings support the idea that DA is a potential therapeutic agent and complementary alternative medicine for the attenuation of trigeminal nociception in the absence of inflammatory/neuropathic conditions.

The electroacupuncture-induced analgesic effect mediated by 5-HT1, 5-HT3 receptor and muscarinic cholinergic receptors in rat model of collagenase-induced osteoarthritis.

BackgroundOsteoarthritis (OA) is an degenerative disease characterized by chronic joint pain. Complementary and alternative treatment such as acupuncture have been utilized to alleviate pain. The objective of this study was to investigate the analgesic mechanisms of electroacupuncture (EA) in the collagenase-induced osteoarthritis (CIOA) rat model.MethodsFour weeks after inducing CIOA by injecting collagenase solution into the left knee of 5-week-old male Sprague-Dawley rats, 2 Hz and 100 Hz EA on Zusanli (ST 36) was performed. The analgesic effect of EA was evaluated by the tail flick latency (TFL) and paw pressure threshold (PPT) tests. To investigate the analgesic mechanism, serotonergic and muscarinic cholinergic receptor agonists and antagonists were injected 20 min prior to EA and the resultant changes were evaluated by the TFL and PPT tests.ResultsEA on Zusanli (ST 36) demonstrated an analgesic effect in the CIOA rat model. The 2 Hz EA treatment showed a significantly greater analgesic effect than the 100 Hz treatment. The analgesic effect of 2 Hz EA was not strengthened by 5-HT1, 5-HT2, 5-HT3, and muscarinic cholinergic receptor agonist pretreatment, was blocked by 5-HT1, 5-HT3, and muscarinic cholinergic receptor antagonist pretreatment, but not blocked by 5-HT2 receptor antagonist pretreatment.ConclusionsIn the CIOA rat model, EA on Zusanli (ST 36) exhibited analgesic effects, and 2 Hz EA resulted in a significantly greater analgesic effect than 100 Hz EA. The analgesic effect of 2 Hz EA was reduced by pretreatment of 5-HT1 receptor, 5-HT3 receptor and muscarinic cholinergic receptor antagonists.

MP60-08 CHRONIC PSYCHOLOGICAL STRESS INDUCES CHANGES IN BLADDER CONTRACTILITY

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology I1 Apr 2016MP60-08 CHRONIC PSYCHOLOGICAL STRESS INDUCES CHANGES IN BLADDER CONTRACTILITY Yunliang Gao, Huiyi Chang, Rong Zhang, and Larissa Rodriguez Yunliang GaoYunliang Gao More articles by this author , Huiyi ChangHuiyi Chang More articles by this author , Rong ZhangRong Zhang More articles by this author , and Larissa RodriguezLarissa Rodriguez More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.861AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chronic psychological stress can impact lower urinary tract function. We aimed to evaluate if the voiding dysfunction and bladder hyperalgesia seen in the animals exposed to water avoidance stress (WAS) is partially due to changes in bladder contractility. METHODS Eight adult female Wistar-Kyoto rats were divided to 10-day WAS protocol (n=4) or handled control (n=4). On day 11, the urinary bladder was harvested, cut into strips, and kept in oxygenated Krebs solution. The remaining of the bladder and urethra were fixed in 10% formalin for histological studies. The contraction amplitude of each strip induced by KCl (80 µM) was regarded as 100% contraction amplitude for normalization. Carbachol, a non-selective cholinergic muscarinic receptor agonist, was given to examine the contractility (10-7 to 3×10-4 M). Atropine (10-6 M), a muscarinic receptor antagonist was given after carbachol to verify the response. Hematoxylin and eosin stain was used to assess tissue structure. RESULTS There were dose dependent responses to carbachol with maximum contractility observed at 10-5 M (Fig 1A). At maximum dose response, there was significantly higher contractility in the bladders of WAS rats than controls (27% higher). Atropine reversed the effect of carbachol on bladder contractility. The thickness and structure of the bladder wall and urethra did not show significantly changes between WAS rats and controls (Fig 1B-C). CONCLUSIONS Chronic psychological stress leads to changes in bladder contractility without changes in the structure of bladder and urethra. Expression or responsiveness of muscarinic receptors induced by stress play a role in the urinary frequency and voiding changes seen in animals exposed to WAS. Receptor characterization will be evaluated in future studies. These results have significant implications in human lower urinary pathologies that have been shown to be exacerbated by affected by stress such as urinary frequency, overactive bladders, and interstitial cystitis. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e796-e797 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yunliang Gao More articles by this author Huiyi Chang More articles by this author Rong Zhang More articles by this author Larissa Rodriguez More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Differential effects of co-administration of oxotremorine with SCH 23390 on impulsive choice in high-impulsive rats and low-impulsive rats

The effect of acetylcholine on impulsive choice is thought to be due to interactions between cholinergic and dopaminergic systems, but this hypothesis has not been proven. This study investigated whether D1-like receptors were involved in the effects of the muscarinic cholinergic agonist oxotremorine on impulsive choice in high-impulsive rats (HI rats, n=8) and low-impulsive rats (LI rats, n=8) characterized by basal levels of impulsive choice in a delay-discounting task. The results revealed that oxotremorine (0.05mg/kg) significantly increased the choice of the large reinforcer in HI rats, whereas decreased the choice of the large reinforcer in LI rats. The D1-like antagonist SCH 23390 produced significant reductions in the large-reinforcer choice in HI rats (0.01mg/kg) and LI rats (0.005, 0.0075, and 0.01mg/kg). SCH 23390 significantly inhibited the increase in the choice of the large reinforcer induced by oxotremorine (0.05mg/kg) in HI rats at doses of 0.005 and 0.0075mg/kg, but enhanced the effect of oxotremorine in LI rats only at the dose of 0.0075mg/kg. These findings suggested that D1-like receptors might be involved in the differential effects of oxotremorine on impulsive choice between HI rats and LI rats.

Retinal ganglion cell neuroprotection induced by activation of alpha7 nicotinic acetylcholine receptors.

Glaucoma is a group of degenerative retinal diseases that damage the eye's optic nerve and can result in vision loss and blindness. It is characterized by optic neuropathy, cupping of the optic disk, and progressive loss of retinal ganglion cells (RGCs) and axons in the optic nerve. Glaucoma patients initially develop loss of visual function in the periphery and slowly develop tunnel vision that becomes worse over time. As the second leading cause of blindness in the world, it is suspected that there are over 60 million cases of glaucoma worldwide. Blindness caused by glaucoma is irreversible. Once adult neurons in the mammalian retina are lost, there is no known treatment that can regenerate new neurons. Although there are known familial genetic causes of glaucoma, there are many more complex cases where the cause is not known. However, the primary risk factor associated with glaucoma is an increase of intraocular pressure (IOP) in the eye and all currently approved treatments are designed to lower IOP. Initial treatments use pharmacological agents to reduce IOP. Topically applied pharmacological treatments include the use of β-blockers, prostaglandins, α-agonists, carbonic anhydrase inhibitors, muscarinic cholinergic agonists or a combination of these. If the use of pharmacological agents to decrease aqueous humor input or decrease aqueous humor outflow is insufficient to reduce IOP, laser therapy or incisional surgery is typically performed. However, these treatments alone can be insufficient to halt the progression of blindness associated with glaucoma. For example, in a subset of patients, RGCs continue to die, even after IOP reduction. In addition, a significant number of glaucoma patients exhibit normal intraocular pressures even though they present typical signs of glaucomatous damage, like optic nerve head excavation and thinning of the retinal nerve fiber layer. Clearly, future glaucoma treatment must involve more than IOP reduction. One avenue of research that addresses this issue involves neuroprotective treatments that work at the level of the retina.

Electrocardiogram, heart movement and heart rate in the awake gecko (Hemidactylus mabouia).

The electrocardiogram (ECG) is the simplest and most effective non-invasive method to assess the electrical activity of the heart and to obtain information on the heart rate (HR) and rhythm. Because information on the HR of very small reptiles (body mass <10 g) is still scarce in the literature, in the present work we describe a procedure for recording the ECG in non-anesthetized geckos (Hemidactylus mabouia, Moreau de Jonnès, 1818) under different conditions, namely manual restraint (MR), spontaneous tonic immobility (TI), and in the non-restrained condition (NR). In the gecko ECG, the P, QRS and T waves were clearly distinguishable. The HR was 2.83 ± 0.02 Hz under MR, which was significantly greater (p < 0.001) than the HR under the TI (1.65 ± 0.09 Hz) and NR (1.60 ± 0.10 Hz) conditions. Spontaneously beating isolated gecko hearts contracted at 0.84 ± 0.03 Hz. The in vitro beating rate was affected in a concentration-dependent fashion by adrenoceptor stimulation with noradrenaline, as well as by the muscarinic cholinergic agonist carbachol, which produced significant positive and negative chronotropic effects, respectively (p < 0.001). To our knowledge, this is the first report on the ECG morphology and HR values in geckos, particularly under TI. The methodology and instrumentation developed here are useful for non-invasive in vivo physiological and pharmacological studies in small reptiles without the need of physical restraint or anesthesia.

Muscarinic presynaptic modulation in GABAergic pallidal synapses of the rat.

The external globus pallidus (GPe) is central for basal ganglia processing. It expresses muscarinic cholinergic receptors and receives cholinergic afferents from the pedunculopontine nuclei (PPN) and other regions. The role of these receptors and afferents is unknown. Muscarinic M1-type receptors are expressed by synapses from striatal projection neurons (SPNs). Because axons from SPNs project to the GPe, one hypothesis is that striatopallidal GABAergic terminals may be modulated by M1 receptors. Alternatively, some M1 receptors may be postsynaptic in some pallidal neurons. Evidence of muscarinic modulation in any of these elements would suggest that cholinergic afferents from the PPN, or other sources, could modulate the function of the GPe. In this study, we show this evidence using striatopallidal slice preparations: after field stimulation in the striatum, the cholinergic muscarinic receptor agonist muscarine significantly reduced the amplitude of inhibitory postsynaptic currents (IPSCs) from synapses that exhibited short-term synaptic facilitation. This inhibition was associated with significant increases in paired-pulse facilitation, and quantal content was proportional to IPSC amplitude. These actions were blocked by atropine, pirenzepine, and mamba toxin-7, suggesting that receptors involved were M1. In addition, we found that some pallidal neurons have functional postsynaptic M1 receptors. Moreover, some evoked IPSCs exhibited short-term depression and a different kind of modulation: they were indirectly modulated by muscarine via the activation of presynaptic cannabinoid CB1 receptors. Thus pallidal synapses presenting distinct forms of short-term plasticity were modulated differently.

Effect of central muscarinic receptors on passive-avoidance learning deficits induced by prenatal pentylenetetrazol kindling in male offspring

Occurrence of the epileptic seizures during gestation might affect the neurodevelopment of the fetus resulting in cognitive problems for the child later in life. We have previously reported that prenatal pentylenetetrazol (PTZ)-kindling induces learning and memory deficits in the children born to kindled mothers, later in life but the mechanisms involved in this processes are unknown. The cholinergic system plays a major role in learning and memory. The present study was performed to investigate the possible involvement of central muscarinic cholinergic receptors on learning and memory deficits induced by prenatal PTZ-kindling in male offspring. Pregnant Wistar rats were kindled by repetitive i.p. injection of 25mg/kg of PTZ on day 13 of their pregnancy. The effect of intracerebroventricular (ICV) microinjection of scopolamine and pilocarpine, muscarinic cholinergic receptors antagonist and agonist, respectively on passive-avoidance learning of pups were tested at 12weeks of age using shuttle-box apparatus. Our data showed that the retention latencies of pups that received scopolamine (2 or 3μg) were significantly reduced compared to those received normal saline (p<0.05). Interestingly, post training ICV administration of pilocarpine (2μg) retrieved pups’ memory deficits (p<0.001).These results demonstrate for the first time, the importance of the central muscarinic cholinergic receptors in learning and memory deficits in pups born to kindled dams and suggest a central mechanism for the cognitive and memory dysfunction, associated with seizures during pregnancy.

The persistence of active smooth muscle in the female rat cervix through pregnancy

A controversy exists as to whether functional smooth muscle exists in the cervix before and during pregnancy, potentially continuous with the uterus. We hypothesized that cervical smooth muscle persists through pregnancy and is functional. Uteri and cervices were taken from female virgin, 11 day, and 20 day (near labor) pregnant rats. All experiments used the uterus as a positive control. Three different smooth muscle proteins (smooth muscle α-actin, SM-22α, and calponin-1) allowed immunohistochemical detection of the continuous nature of the smooth muscle from the vagina, cervix, and uterus. Tissues were also hung in isolated tissue baths for the measurement of isometric smooth muscle contraction. Uterine and cervical homogenates were also used in Western analyses to measure protein expression. Immunohistochemistry revealed there to be smooth muscle as validated by an expression of all 3 markers in the cervix. This smooth muscle was continuous with that of the vagina and uterus. Smooth muscle α-actin was detected in virgin tissue (291.3 ± 32.2 arbitrary densitometry units/β-actin), day 11 (416.8 ± 19.5), and day 20 pregnant tissue (293.0 ± 34.4). The virgin, day 11, and day 20 cervices contracted 2.18 ± 0.24 g, 1.46 ± 0.08 g, and 3.88 ± 0.49 g (respectively) to depolarizing KCl. Cervices contracted at day 20 to the cholinergic muscarinic agonist carbamylcholine (maximum, 133% ± 18.2% KCl contraction, n = 4). These findings strongly support that smooth muscle is present in the cervix through pregnancy and continuous with the uterus.

Muscarinic stimulation and pinacidil produce similar facilitation of tachyarrhythmia induction in rat isolated atria

Atrial tachyarrhythmias, the most common type of cardiac arrhythmias, are associated with greater stroke risk. Muscarinic cholinergic agonists have been shown to facilitate atrial tachyarrhythmia maintenance in the absence of cardiac disease. This has been attributed to action potential shortening, which enhances myocardial electrical anisotropy, and thus creates a substrate for reentrant excitation. In this study, we describe a similar effect of the ATP-sensitive K+ channel (KATP) opener pinacidil on tachyarrhythmia induction in isolated rat atria. Pinacidil, which activates a weakly inwardly-rectifying current in isolated atrial myocytes, enhanced arrhythmia induction in the right and left atria. This effect was abolished by the KATP blocker glibenclamide, but not by atropine, which rules out a possible indirect effect due to stimulation of acetylcholine release. However, pinacidil attenuated carbachol-induced tachyarrhythmia facilitation, which may indicate that the action of these agonists converges to a common cellular mechanism. Both agonists caused marked action potential shortening in isolated atrial myocytes. Moreover, during arrhythmia in the presence of pinacidil and carbachol, the atrial vectorelectrographic patterns were similar and consistent with reentrant propagation of the electrical activity. From these results, we conclude that the KATP channel opening is pro-arrhythmic in atrial tissue, which may pose as an additional risk in the scenario of myocardial hypoxia. Moreover, the similarity of the electrophysiological effects of pinacidil and carbachol is suggestive that the sole increase in background K+ conductance is sufficient for atrial tachyarrhythmia facilitation.

Propranolol prevents life-threatening arrhythmias in LQT3 transgenic mice: Implications for the clinical management of LQT3 patients

BackgroundThe efficacy of beta-blockers for treatment of patients with long QT syndrome type 3 (LQT3) has been repeatedly questioned, and it has been suggested that they might be detrimental for this genetic subgroup of patients with long QT syndrome (LQTS). The disquieting consequence has been that cardiologists confronted with LQT3 patients often do not even attempt pharmacologic therapy and implant cardioverter-defibrillators as first-choice treatment. However, the most recent clinical data indicate high efficacy of beta-blocker therapy in LQT3 patients.ObjectiveThe purpose of this study was to test the antiarrhythmic efficacy of beta-blockers in an established experimental model for LQT3.MethodsAfter phenotypic validation of 65 ∆KPQ-SCN5A knock-in transgenic (TG) mice compared to 32 wild-type (WT) mice, we tested the effect of the arrhythmogenic cholinergic muscarinic agonist carbachol in 19 WT and 39 TG anesthetized mice, with and without pretreatment with propranolol given intraperitoneally.ResultsAt the same heart rates, TG mice had a markedly longer QT interval than WT mice. Whereas carbachol had minor arrhythmic effects in the WT mice, it produced ventricular tachycardia (VT) and ventricular fibrillation (VF) in 55% of 20 TG mice. By contrast, in none of 19 TG mice pretreated with propranolol did VT/VF occur after carbachol injection.ConclusionThese experimental data indicate that, contrary to previous reports, beta-blockade effectively prevents VT/VF in a validated LQT3 model. Together with the most recent clinical data, these findings indicate that there is no reason for not initiating protective therapy with beta-blockers in LQT3 patients.

GABAA receptors implicated in REM sleep control express a benzodiazepine binding site

It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10−2M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

Cholinergic modulation of olfactory pattern separation

Pattern separation plays an important role in perception and memory. In olfaction, pattern separation is critical component of piriform cortical odor processing contributing to behavioral perception of overlapping odor mixtures. Previous work has demonstrated that odor discrimination ability is modulated by acetylcholine. Here, we extended this previous work by using a distinct, well characterized complex odor stimulus set that has been shown to differentially involve pattern separation processes within piriform cortex. We find that the cholinergic muscarinic receptor agonist oxotremorine facilitates the acquisition of odor discrimination. Furthermore, the muscarinic receptor antagonist scopolamine impairs acquisition of odor discrimination even if the antagonist is limited to the piriform cortex. Finally, acetylcholine effects are most robust during discrimination acquisition, with minimal effects during expression.

H2S Is an Endothelium-Derived Hyperpolarizing Factor

Endothelium-dependent vasorelaxation is mediated by endothelium-derived relaxing factor and endothelium-derived hyperpolarizing factor (EDHF). However, the molecular entity of EDHF remains unclear. The present study examined whether hydrogen sulfide (H₂S) acts as EDHF and how H₂S mediates EDHF pathways from endothelial origination to downstream target of smooth muscle cells (SMCs). We found that knocking-out the expression of cystathionine γ-lyase (CSE) in mice (CSE-knockout [KO]) elevated resting-membrane-potential of SMCs and eliminated methacholine-induced endothelium-dependent relaxation of mesenteric arteries, but not that of aorta. Methacholine, a cholinergic-muscarinic agonist, hyperpolarized SMC in endothelium-intact mesenteric arteries from wide-type mice. This effect was inhibited by muscarinic antagonist (atropine) or the co-application of charybdotoxin and apamin, which blocked intermediate- and small-conductance KCa (IKCa and SKCa) channels, or abolished in CSE-KO mice. Supplementation of exogenous H₂S hyperpolarized vascular SMCs and endothelial cells from wide-type and CSE-KO mice. Both methacholine and H₂S induced greater SMC hyperpolarization of female wide-type mesenteric arteries than that of male ones. H2S-induced hyperpolarization is blocked by -SH oxidants and -SSH inhibitor. The expression of SK2.3 but not IK3.1 channel in vascular tissues was increased by H₂S and decreased by CSE inhibitor or CSE gene KO. Taken together, H₂S is an EDHF. The identification of H2S as an EDHF will not only solve one of the long-lasting perplexing puzzles for the mechanisms underlying endothelium-dependent vasorelaxation, but also shed light on potential therapeutic effects of H₂S on pathological abnormalities in peripheral resistance arteries.

Effect of sialagogue on bleeding on probing in Sjögren′s syndrome

Bleeding on probing (BOP) is a frequent observation in patients with Sjögren's syndrome and a sialagogue is routinely prescribed for these patients. The objective of this study was to evaluate the effect of sialagogue (muscarinic cholinergic agonists) on BOP in patients with Sjögren's syndrome. This observational study included 57 subjects. Study population was divided into two groups: Subjects on sialagogue (n = 32) and subjects not on sialagogue due to their side-effects (non-sialagogue, n = 25). The number of sites with BOP was recorded on all teeth. The subjects on sialagogue had a significantly lower mean (standard error) number of sites with BOP 22.97 (2.65) as compared with the non-sialagogue group 46.59 (6.20), P < 0.001. After adjusting for the use of remineralizing rinse the subjects on sialagogue had a significantly lower number of sites with BOP (P < 0.001). In this observational study treatment with sialagogue may prevent BOP in patients with Sjögren's syndrome.

Effects of acute administration of nicotinic and muscarinic cholinergic agonists and antagonists on performance in different cost–benefit decision making tasks in rats

Alterations in cost-benefit decision making accompany numerous neuropsychiatric conditions, including schizophrenia, attention deficit hyperactivity disorder, and addiction. Central cholinergic systems have been linked to the etiology and/or treatment of many of these conditions, but little is known about the role of cholinergic signaling in cost-benefit decision making. The goal of these experiments was to determine how cholinergic signaling is involved in cost-benefit decision making, using a behavioral pharmacological approach. Male Long-Evans rats were trained in either "probability discounting" or "delay discounting" tasks, in which rats made discrete-trial choices between a small food reward and a large food reward associated with either varying probabilities of omission or varying delays to delivery, respectively. The effects of acute administration of different doses of nicotinic and muscarinic acetylcholine receptor agonists and antagonists were assessed in each task. In the probability discounting task, acute nicotine administration (1.0mg/kg) significantly increased choice of the large risky reward, and control experiments suggested that this was due to robust nicotine-induced impairments in behavioral flexibility. In the delay discounting task, the muscarinic antagonists scopolamine (0.03, 0.1, and 0.3mg/kg) and atropine (0.3mg/kg) both significantly increased choice of the small immediate reward. Neither mecamylamine nor oxotremorine produced reliable effects on either of the decision making tasks. These data suggest that cholinergic receptors play multiple roles in decision making contexts which include consideration of reward delay or probability. These roles should be considered when targeting these receptors for therapeutic purposes.

Smooth Muscle Pharmacology in the Isolated Virgin and Pregnant Rat Uterus and Cervix

Uterine smooth muscle function is established, but comparatively little is known about cervical smooth muscle pharmacology. We performed a proof-of-principle experiment that smooth muscle was expressed in the cervix in both virgin and pregnant rats, using the uterus as a comparator. We tested whether all tissues were pharmacologically responsive to contractile and relaxant agonists. Immunohistochemistry revealed the expression of smooth muscle α-actin in all tissues. The isolated tissue bath was used to measure isometric contractility of uterine strips and whole cervices from virgin and pregnant (day 11 ± 2) female Sprague-Dawley rats. We tested classic activators of uterine smooth muscle contraction and relaxation in both uterus and cervix. All tissues contracted to the depolarizing agent potassium chloride, prostaglandin F2α, muscarinic cholinergic agonist carbachol [2-[(aminocarbonxyl)oxy]-N,N,N-trimethylethanaminium chloride], and 5-hydroxytryptamine. Unlike other tissues, the pregnant cervix did not contract to oxytocin, but the oxytocin receptor was present. Both cervix and uterus (virgin and pregnant) had concentration-dependent, near-complete relaxation to the adrenergic agonist norepinephrine and adenylate cyclase activator forskolin [(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-6,10-10b-trihydroxy-3,4a,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f] chroment-5-yl acetate]. The β-adrenergic receptor agonist isoproterenol was less potent in pregnant cervix versus virgin by ∼10-fold. All tissues, particularly the cervix, responded poorly to the nitric-oxide donor sodium nitroprusside, relaxing ∼20% maximally. These findings support the importance of smooth muscle in the cervix, the use of the isolated cervix in pharmacological studies, and a similarity between smooth muscle pharmacology of the nonpregnant uterus and cervix. This work highlights the unappreciated smooth muscle function of the cervix versus uterus and cervical changes in pharmacology during pregnancy.

Effects of Autonomic Agents on Ca2+ Cycling in Canine Atrial Myocytes during Rapid Pacing

Atrial fibrillation (AF), or conditions conducive to it, often occur(s) in conjunction with high and/or unbalanced sympathetic/parasympathetic (autonomic) activity, which has profound effects on myocyte Ca2+ cycling. Using confocal microscopy, we have begun scrutinizing the effects various autonomic agents–including but not limited to isoproterenol (ISO; ∗-adrenergic receptor (AR) agonist), phenylephrine (PE; ∗-AR agonist) norepinephrine (NE, ∗- and ∗-AR agonist), and carbachol (CCh; muscarinic cholinergic receptor agonist)–have on Ca2+ cycling in isolated canine atrial myocytes paced at cycle lengths (CLs) ranging from 5000-200ms. In general, Ca-transient amplitudes were increased by ISO and NE; decreased by CCh; and varying decreased, increased or unaffected by PE–although PE only increased Ca-transients after PTX-treatment. However, considerable cell-to-cell variability in magnitude/dose-response for such effects was notable. The effects these agents had on irregular Ca-release events (ICREs)–i.e., Ca-alternans triggered-Ca-waves occurring during pacing (t-CaWs), and spontaneous-Ca-waves occurring during a pause after pacing (s-CaWs)–consequent to rapid pacing (CLs≤300ms) were interestingly distinct. CCh significantly reduced the appearance of all ICREs, yet were often accentuated upon CCh withdrawal. ISO often induced s-CaWs, but suppressed t-CaWs–with Ca-alternans often appearing in their stead. NE mimicked ISO regarding ICREs in some cells, but in others did not suppress and sometimes accentuated t-CaWs. PE accentuating t-CaWs and/or subcellular Ca-alternans in some cells, while suppressing or having no apparent effect on them in others. However, after PTX-treatment, PE mimicked ISO regarding ICREs without s-CaWs induction. These findings not only underscore the complexity of atrial autonomic modulation and its differences with that in ventricle, but also particulars evident only during rapid pacing–i.e., that during or conducive to the onset of AF.

Mapping a mouse limbic seizure susceptibility locus on chromosome 10

Mapping seizure susceptibility loci in mice provides a framework for identifying potentially novel candidate genes for human epilepsy. Using C57BL/6J × A/J chromosome substitution strains (CSS), we previously identified a locus on mouse chromosome 10 (Ch10) conferring susceptibility to pilocarpine, a muscarinic cholinergic agonist that models human temporal lobe epilepsy by inducing initial limbic seizures and status epilepticus (status), followed by hippocampal cell loss and delayed-onset chronic spontaneous limbic seizures. Herein we report further genetic mapping of pilocarpine quantitative trait loci (QTLs) on Ch10. Seventy-nine Ch10 F(2) mice were used to map QTLs for duration of partial status epilepticus and the highest stage reached in response to pilocarpine. Based on those results we created interval-specific congenic lines to confirm and extend the results, using sequential rounds of breeding selectively by genotype to isolate segments of A/J Ch10 genome on a B6 background. Analysis of Ch10 F(2) genotypes and seizure susceptibility phenotypes identified significant, overlapping QTLs for duration of partial status and severity of pilocarpine-induced seizures on distal Ch10. Interval-specific Ch10 congenics containing the susceptibility locus on distal Ch10 also demonstrated susceptibility to pilocarpine-induced seizures, confirming results from the F(2) mapping population and strongly supporting the presence of a QTL between rs13480781 (117.6 Mb) and rs13480832 (127.7 Mb). QTL mapping can identify loci that make a quantitative contribution to a trait, and eventually identify the causative DNA-sequence polymorphisms. We have mapped a locus on mouse Ch10 for pilocarpine-induced limbic seizures. Novel candidate genes identified in mice can be investigated in functional studies and tested for their role in human epilepsy.

Central mechanisms involved in pilocarpine-induced pressor response

Pilocarpine (cholinergic muscarinic agonist) injected peripherally may act centrally to produce pressor responses; in the present study, using c-fos immunoreactive expression, we investigated the forebrain and brainstem areas activated by pressor doses of intravenous (i.v.) pilocarpine. In addition, the importance of vasopressin secretion and/or sympathetic activation and the effects of lesions in the anteroventral third ventricle (AV3V) region in awake rats were also investigated. In male Holtzman rats, pilocarpine (0.04 to 4μmol/kg b.w.) i.v. induced transitory hypotension followed by long lasting hypertension. Sympathetic blockade with prazosin (1mg/kg b.w.) i.v. or AV3V lesions (1day) almost abolished the pressor response to i.v. pilocarpine (2μmol/kg b.w.), whereas the vasopressin antagonist (10μg/kg b.w.) i.v. reduced the response to pilocarpine. Pilocarpine (2 and 4μmol/kg b.w.) i.v. increased the number of c-fos immunoreactive cells in the subfornical organ, paraventricular and supraoptic nuclei of the hypothalamus, organ vasculosum of the lamina terminalis, median preoptic nucleus, nucleus of the solitary tract and caudal and rostral ventrolateral medulla. These data suggest that i.v. pilocarpine activates specific forebrain and brainstem mechanisms increasing sympathetic activity and vasopressin secretion to induce pressor response.