Sex-differences in cholinergic, nicotinic, and ß-adrenergic cutaneous vasodilation: Roles of nitric oxide synthase, cyclooxygenase, and K+ channels.

Previous studies indicate that sex-related differences exist in the regulation of cutaneous vasodilation, however, the mechanisms remain unresolved. We assessed if sex-differences in young adults exist for cholinergic, nicotinic, and ß-adrenergic cutaneous vasodilation with a focus on nitric oxide synthase (NOS), cyclooxygenase (COX), and K+ channel mechanisms. In twelve young men and thirteen young women, four intradermal forearm skin sites were perfused with the following: 1) lactated Ringer's solution (control), 2) 10 mM Nω-nitro-l-arginine, a non-selective NOS inhibitor, 3) 10 mM ketorolac, a non-selective COX inhibitor, or 4) 50 mM BaCl2, a nonspecific K+ channel blocker. At all four sites, cutaneous vasodilation was induced by 1) 10 mM nicotine, a nicotinic receptor agonist, 2) 100 µM isoproterenol, a nonselective ß-adrenergic receptor agonist, and 3) 2 mM and 2000 mM acetylcholine, an acetylcholine receptor agonist. Nicotine and isoproterenol were administered for 3 min, whereas each acetylcholine dose was administered for 25 min. Regardless of treatment site, cutaneous vasodilation in response to nicotine and a high dose of acetylcholine (2000 mM) were lower in women than men. By contrast, isoproterenol induced cutaneous vasodilation was greater in women vs. men. Irrespective of sex, NOS inhibition or K+ channel blockade attenuated isoproterenol-mediated cutaneous vasodilation, whereas K+ channel blockade decreased nicotine-induced cutaneous vasodilation. Taken together, our findings indicate that while the mechanisms underlying cutaneous vasodilation are comparable between young men and women, sex-related differences in the magnitude of cutaneous vasodilation do exist and this response differs as a function of the receptor agonist.

Acetylcholine acts on songbird premotor circuitry to invigorate vocal output.

Acetylcholine is well-understood to enhance cortical sensory responses and perceptual sensitivity in aroused or attentive states. Yet little is known about cholinergic influences on motor cortical regions. Here we use the quantifiable nature of birdsong to investigate how acetylcholine modulates the cortical (pallial) premotor nucleus HVC and shapes vocal output. We found that dialyzing the cholinergic agonist carbachol into HVC increased the pitch, amplitude, tempo and stereotypy of song, similar to the natural invigoration of song that occurs when males direct their songs to females. These carbachol-induced effects were associated with increased neural activity in HVC and occurred independently of basal ganglia circuitry. Moreover, we discovered that the normal invigoration of female-directed song was also accompanied by increased HVC activity and was attenuated by blocking muscarinic acetylcholine receptors. These results indicate that, analogous to its influence on sensory systems, acetylcholine can act directly on cortical premotor circuitry to adaptively shape behavior.

Muscarinic and Nicotinic Modulation of Neocortical Layer 6A Synaptic Microcircuits Is Cooperative and Cell-Specific.

Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, for example, wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M 4 and M1 mAChRs, respectively. At ~ 1 mM, ACh depolarized exclusively CT PCs via α4ß2 subunit-containing nAChRs without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was suppressed via M4 mAChR activation but enhanced by nAChRs via α4ß2 nAChRs when the presynaptic neuron was a CT PC. Thus, in L6A, the interaction of mAChRs and nAChRs results in an altered excitability and synaptic release, effectively strengthening CT output while weakening CC synaptic signaling.

Berberine for prevention of dementia associated with diabetes and its comorbidities: A systematic review.

BACKGROUND: A growing number of epidemiological studies indicate that metabolic syndrome (MetS) and its associated features play a key role in the development of certain degenerative brain disorders, including Alzheimer's disease and vascular dementia. Produced by several different medicinal plants, berberine is a bioactive alkaloid with a wide range of pharmacological effects, including antidiabetic effects. However, it is not clear whether berberine could prevent the development of dementia in association with diabetes. OBJECTIVE: To give an overview of the therapeutic potential of berberine as a treatment for dementia associated with diabetes. SEARCH STRATEGY: Database searches A and B were conducted using PubMed and ScienceDirect. In search A, studies on berberine's antidementia activities were identified using "berberine" and "dementia" as search terms. In search B, recent studies on berberine's effects on diabetes were surveyed using "berberine" and "diabetes" as search terms. INCLUSION CRITERIA: Clinical and preclinical studies that investigated berberine's effects associated with MetS and cognitive dysfunction were included. DATA EXTRACTION AND ANALYSIS: Data from studies were extracted by one author, and checked by a second; quality assessments were performed independently by two authors. RESULTS: In search A, 61 articles were identified, and 22 original research articles were selected. In search B, 458 articles were identified, of which 101 were deemed relevant and selected. Three duplicates were removed, and a total of 120 articles were reviewed for this study. The results demonstrate that berberine exerts beneficial effects directly in the brain: enhancing cholinergic neurotransmission, improving cerebral blood flow, protecting neurons from inflammation, limiting hyperphosphorylation of tau and facilitating ß-amyloid peptide clearance. In addition, evidence is growing that berberine is effective against diabetes and associated disorders, such as atherosclerosis, cardiomyopathy, hypertension, hepatic steatosis, diabetic nephropathy, gut dysbiosis, retinopathy and neuropathy, suggesting indirect benefits for the prevention of dementia. CONCLUSION: Berberine could impede the development of dementia via multiple mechanisms: preventing brain damages and enhancing cognition directly in the brain, and indirectly through alleviating risk factors such as metabolic dysfunction, and cardiovascular, kidney and liver diseases. This study provided evidence to support the value of berberine in the prevention of dementia associated with MetS.

Effect of Atropine on Adrenergic Responsiveness of Erythrocyte and Heart Rhythm Variability in Outbred Rats with Stimulation of the Central Neurotransmitter Systems.

Single injection of muscarinic cholinoceptor blocker atropine (1 mg/kg) to outbred male rats reduced ß-adrenergic responsiveness of erythrocytes (by 2.2 times) and the content of epinephrine granules on erythrocytes (by 1.5 times), significantly increased HR and rigidity of the heart rhythm, and manifold decreased the power of all spectral components of heart rhythm variability. Stimulation of the central neurotransmitter systems increased ß-adrenergic responsiveness of erythrocytes (by 15-26%), decreased the number of epinephrine granules on erythrocytes (by 25-40%), and increased HR and cardiac rhythm intensity. These changes were most pronounced after stimulation of the serotoninergic system. Administration of atropine against the background of activation of central neurotransmitter systems did not decrease ß-adrenergic responsiveness of erythrocytes (this parameter remained at a stably high level and even increased during stimulation of the dopaminergic system), but decreased the number of epinephrine granules on erythrocytes, increased HR, and dramatically decreased the power of all components of heart rhythm variability spectrum. The response to atropine was maximum against the background of noradrenergic system activation and less pronounced during stimulation of the serotoninergic system. Thus, substances that are complementary to cholinergic receptors modulated adrenergic effect on the properties of red blood cells, which, in turn, can modulate the adrenergic influences on the heart rhythm via the humoral channel of regulation. Stimulation of central neurotransmitter systems that potentiates the growth of visceral adrenergic responsiveness weakens the cholinergic modulation of the adrenergic influences, especially with respect to erythrocyte responsiveness. Hence, changes in the neurotransmitter metabolism in the body can lead to coupled modulation of reception and reactivity to adrenergic- and choline-like regulatory factors at the level of erythrocyte membranes, which can be important for regulation of heart rhythm.

Cre-expressing neurons in visual cortex of Ntsr1-Cre GN220 mice are corticothalamic and are depolarized by acetylcholine.

The Ntsr1-Cre GN220 mouse expresses Cre-recombinase in corticothalamic (CT) neurons in neocortical layer 6. It is not known if the other major types of pyramidal neurons in this layer also express this enzyme. By electrophysiological recordings in slices and histological analysis of the uptake of retrogradely transported beads we show that Cre-positive neurons are CT and not corticocortical or corticoclaustral types. Furthermore, we show that Ntsr1-Cre-positive cells are immuno-positive for the nuclear transcription factor Forkhead box protein P2 (FoxP2). We conclude that Cre-expression is limited to a specific type of pyramidal neuron: CT. However, it appears as not all CT neurons are Cre-expressing; there are indications that the penetrance of the gene is about 90%. We demonstrate the utility of assigning a specific identity to individual neurons by determining that the CT neurons are potently modulated by acetylcholine acting on both nicotinic and muscarinic acetylcholine receptors. These results corroborate the suggested function of these neurons in regulating the gain of thalamocortical transfer of sensory information depending on attentional demand and state of arousal.

Fucoidan inhibits Ca2+ responses induced by a wide spectrum of agonists for G­protein­coupled receptors.

Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has been used in traditional Chinese herbal medicine to treat thyroid tumors for many years. Although a number of its cellular effects have been investigated, the role of fucoidan in molecular signaling, particularly in Ca2+ signaling, remains largely unknown. In the present study, the effects of fucoidan on Ca2+ responses in HeLa cells, human umbilical vein endothelial cells and astrocytes were investigated using a wide range of receptor agonists. Fucoidan inhibited the increase in intracellular free calcium concentration that was induced by histamine, ATP, compound 48/80 and acetylcholine. The responses induced by the same agonists in the absence of extracellular Ca2+ were also markedly suppressed by fucoidan. Reverse transcription­polymerase chain reaction demonstrated that 0.5 and 1.0 mg/ml fucoidan treatment for 3 h decreased histamine receptor 1 expression in HeLa cells. Similarly, the expressions of purinergic receptor P2Y, G­protein coupled (P2YR)1, P2YR2 and P2YR11 were significantly downregulated within cells pretreated with 1.0 mg/ml fucoidan for 3 h, and 0.5 mg/ml fucoidan significantly inhibited P2YR1 and P2YR11 expression. The results demonstrated that fucoidan may exert a wide spectrum of inhibitory effects on Ca2+ responses and that fucoidan may inhibit a number of different G­protein coupled receptors associated with Ca2+ dynamics.

Phase-amplitude coupled persistent theta and gamma oscillations in rat primary motor cortex in vitro.

In vivo, theta (4-7 Hz) and gamma (30-80 Hz) neuronal network oscillations are known to coexist and display phase-amplitude coupling (PAC). However, in vitro, these oscillations have for many years been studied in isolation. Using an improved brain slice preparation technique we have, using co-application of carbachol (10 µM) and kainic acid (150 nM), elicited simultaneous theta (6.6 ± 0.1 Hz) and gamma (36.6 ± 0.4 Hz) oscillations in rodent primary motor cortex (M1). Each oscillation showed greatest power in layer V. Using a variety of time series analyses we detected significant cross-frequency coupling in 74% of slice preparations. Differences were observed in the pharmacological profile of each oscillation. Thus, gamma oscillations were reduced by the GABAA receptor antagonists, gabazine (250 nM and 2 µM), and picrotoxin (50 µM) and augmented by AMPA receptor antagonism with SYM2206 (20 µM). In contrast, theta oscillatory power was increased by gabazine, picrotoxin and SYM2206. GABAB receptor blockade with CGP55845 (5 µM) increased both theta and gamma power, and similar effects were seen with diazepam, zolpidem, MK801 and a series of metabotropic glutamate receptor antagonists. Oscillatory activity at both frequencies was reduced by the gap junction blocker carbenoxolone (200 µM) and by atropine (5 µM). These data show theta and gamma oscillations in layer V of rat M1 in vitro are cross-frequency coupled, and are mechanistically distinct. The development of an in vitro model of phase-amplitude coupled oscillations will facilitate further mechanistic investigation of the generation and modulation of coupled activity in mammalian cortex.

Developmental nicotine exposure alters cholinergic control of respiratory frequency in neonatal rats.

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem-spinal cord preparation in the split-bath configuration, and applied drugs to the brainstem compartment while measuring the burst frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: (1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; (2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; (3) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; (4) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1138-1149, 2016.

Effects of papaverine on carbachol- and high K+ -induced contraction in the bovine abomasum.

The effects of papaverine on carbachol (CCh) -and high K(+)- induced contraction in the bovine abomasum were investigated. Papaverine inhibited CCh (1 µM) -and KCl (65 mM) -induced contractions in a concentration-dependent manner. Forskolin or sodium nitroprusside inhibited CCh-induced contractions in a concentration-dependent manner in association with increases in the cAMP or cGMP contents, whereas papaverine increased cGMP contents only at 30 µM. Changes in the extracellular Ca(2+) from 1.5 mM to 7.5 mM reduced verapamil-induced relaxation in high K(+)-depolarized muscles, but papaverine-induced relaxation did not change. Furthermore, papaverine (30 µM) and NaCN (300 µM) decreased the creatine phosphate contents. These results suggest that the relaxing effects of papaverine on the bovine abomasum are mainly due to the inhibition of aerobic energy metabolism.

Interaction of aqueous leaf extract of Aegle marmelos (L.) Corr. with cholinergic, serotonergic and adrenergic receptors: an ex vivo study.

OBJECTIVES: The aim was to study interaction of aqueous leaf extract of Aegle marmelos (AM) with cholinergic, serotonergic, and adrenergic receptor systems using appropriate rat tissues-ileum, fundus and tracheal chain, respectively. MATERIALS AND METHODS: Cumulative concentration-response curves (CRC) were constructed at various doses on each tissue for AM and respective standard agonist. The CRC was again plotted in presence and absence of respective standard antagonist to confirm the interaction of receptor system and AM. RESULTS: AM induced concentration-dependent contractions in isolated rat ileum (0.2-6.4 mg/ml) and fundus (0.2-3.2 mg/ml) that were inhibited significantly (P < 0.05) in the presence of atropine (10(-7) M) and ketanserin (10(-6) M), respectively. The relaxant effect, produced by AM (0.2 mg/ml) on carbachol (10(-5) M) precontracted rat tracheal chain, was also inhibited significantly (P < 0.05) by propranolol (1 ng/ml). CONCLUSION: It may be concluded that AM possesses agonistic activity on cholinergic, serotonergic and adrenergic receptors.

Wake-sleep, thermoregulatory, and autonomic effects of cholinergic activation of the lateral hypothalamus in the rat: a pilot study.

A major role in the wake-promoting effects of the activation of the lateral hypothalamus (LH) has been ascribed to a population of orexin (ORX)-containing neurons that send projections to central areas which regulate Wake-Sleep and autonomic function. Since, in the rat, a substantial amount of ORX neurons receive cholinergic projections from cells involved in Wake-Sleep regulation, the aim of this study was to assess the role played by LH cholinoceptive cells in Wake-Sleep and autonomic regulations. To this end, the effects of a microinjection of the cholinergic agonist Carbachol (CBL) into the LH were compared to those obtained through the activation of a wider cell population by the microinjection of the GABAA antagonist GABAzine (GBZ). The results of this pilot study showed that both drugs elicited the same behavioral and autonomic effects, those caused by GBZ being larger and longer-lasting than those following administration of CBL. Briefly, wakefulness was enhanced and sleep was depressed, and brain temperature and heart rate consistently increased, while mean arterial pressure showed only a mild increment. Surprisingly, the administration of the drug vehicle (SAL) elicited a similar pattern of Wake-Sleep effects which, although much smaller, were sufficient to mask any statistical significance between treatment and control data. In conclusion, the results of this work show that the arousal elicited by LH disinhibition by GABAzine is concomitant with autonomic responses set by the intervention of cold-defense mechanisms. Since the same response is elicited at a lower level by CBL administration, the hypothesis of an involvement of cholinoceptive ORX neurons in its generation is discussed.

Izalpinin from fruits of Alpinia oxyphylla with antagonistic activity against the rat bladder contractility.

BACKGROUND: Alpinia oxyphylla (Zingiberaceae), an herbaceous perennial plant, its capsular fruit is commonly used in traditional Chinese medicine for the treatment of different urinary incontinence symptoms including frequency, urgency and nocturia. These symptoms are similar to the overactive bladder syndrome. In our lab, we found that the 95% ethanol extract of the capsular fruits exhibited significant anti-muscarinic activity. Some constituents in capsular fruits including flavonoids (e.g., izalpinin and tectochrysin), diarylheptanoids (e.g., yakuchinone A and yakuchinone B) and sesquiterpenes (e.g., nootkatone), are regarded as representative chemicals with putative pharmacological activities. OBJECTIVE: This study aimed to evaluate the in vitro antagonistic actions of izalpinin on carbachol-induced contraction of the rat detrusor muscle. MATERIALS AND METHODS: In vitro inhibition of rat detrusor contractile response to carbachol was used to study the functional activity of izalpinin. The isolated detrusor strips of rats were mounted in organ baths containing oxygenated Krebs' solution. The cumulative consecutive concentration-response curves to carbachol-evoked contractions in strips of rat bladder were obtained. RESULTS: Carbachol induced concentration-dependent contractions of isolated rat bladder detrusor strips. The vehicle DMSO had no impact on the contraction response. The contraction effects were concentration-dependently antagonized by izalpinin, with a mean EC50 value of 0.35 µM. The corresponding cumulative agonist concentration-response curves shifted right-ward. CONCLUSIONS: Izalpinin exhibits inhibitory role of muscarinic receptor-related detrusor contractile activity, and it may be a promising lead compound to treat overactive bladder.

Cardiac contractility modulation increases action potential duration dispersion and decreases ventricular fibrillation threshold via ß1-adrenoceptor activation in the crystalloid perfused normal rabbit heart.

BACKGROUND/OBJECTIVES: Cardiac contractility modulation (CCM) is a new treatment being developed for heart failure (HF) involving application of electrical current during the absolute refractory period. We have previously shown that CCM increases ventricular force through ß1-adrenoceptor activation in the whole heart, a potential pro-arrhythmic mechanism. This study aimed to investigate the effect of CCM on ventricular fibrillation susceptibility. METHODS: Experiments were conducted in isolated New Zealand white rabbit hearts (2.0-2.5 kg, n=25). The effects of CCM (± 20 mA, 10 ms phase duration) on the left ventricular basal and apical monophasic action potential duration (MAPD) were assessed during constant pacing (200 bpm). Ventricular fibrillation threshold (VFT) was defined as the minimum current required to induce sustained VF with rapid pacing (30 × 30 ms). Protocols were repeated during perfusion of the ß1-adrenoceptor antagonist metoprolol (1.8 µM). In separate hearts, the dynamic and spatial electrophysiological effects of CCM were assessed using optical mapping with di-4-ANEPPS. RESULTS: CCM significantly shortened MAPD close to the stimulation site (Basal: 102 ± 5 [CCM] vs. 131 ± 6 [Control] ms, P<0.001). VFT was reduced during CCM (2.6 ± 0.6 [CCM] vs. 6.1 ± 0.8 [Control] mA, P<0.01) and was correlated (r(2)=0.40, P<0.01) with increased MAPD dispersion (26 ± 4 [CCM] vs. 5 ± 1 [Control] ms, P<0.01) (n=8). Optical mapping revealed greater spread of CCM induced MAPD shortening during basal vs. apical stimulation. CCM effects were abolished by metoprolol and exogenous acetylcholine. No evidence for direct electrotonic modulation of APD was found, with APD adaptation occurring secondary to adrenergic stimulation. CONCLUSIONS: CCM decreases VFT in a manner associated with increased MAPD dispersion in the crystalloid perfused normal rabbit heart.

Effects of Ammi visnaga (Bisnaga) extract on the volume and acidity of stimulated gastric secretion in fasting rabbits.

OBJECTIVE: To determine the efficacy of extract of Ammi visnaga on volume and acidity of stimulated gastric secretion in rabbits and also its safety on liver and kidney function. STUDY DESIGN: Quasi experimental study. PLACE AND DURATION OF STUDY: Pharmacology Department, Saidu Medical College, Swat, in the years 2008-9. METHODOLOGY: Thirty rabbits weighing 1 - 1.5 kg were divided into groups A, B and C each having 10 animals. After fasting for 48 hours, pylorus of animals of group A and B was ligated. Group A was administered Carbachol and group B was given extracts of Ammi visnaga followed by Carbachol after 15 minutes intraperitoneally. After 4 hours, stomach contents were measured for volume and then centrifuged and estimated for acidity. The extract was also administered to group C animals for 45 days to observe its effects on liver and kidney function. RESULTS: In group B, reduction in volume, free and total acidity of gastric juice was highly significant when the mean values were compared with group A. In group C, mean values of liver and kidney function test compared with pre-treated values, were found statistically non-significant. CONCLUSION: Ammi visnaga extract can be used effectively and safely in the treatment of hyper acidity conditions and peptic ulcer after evaluation of its effects in human being.

Repetitive administration of acetylcholine receptor agonist rescues brain inflammation and brain damage after hypoxia-ischemia in newborn rat.

OBJECTIVE: We examined the effect of repetitive administration of acetylcholine receptor agonist (carbachol) on brain damage and microglial accumulation in three brain regions after hypoxia-ischemia (HI) in newborn rat. STUDY DESIGN: Seven-day-old Wistar rats were divided into two groups, one receiving a 0.1 mg/kg dose of carbachol on days 7, 8 and 9 to examine the attenuating effect on brain damage with decreasing accumulation of microglia, and the other group receiving saline as a control. Rats were subjected to left carotid artery ligation followed by hypoxia. We evaluated brain damage and the number of microglias in three regions on days 10 and 14. RESULTS: Brain tissue was better preserved in the carbachol group on days 10 and 14. Microglial accumulation in the cortex was strong and persisted from day 10s to 14 in the control. Conversely, the accumulation of microglias was attenuated in the hippocampus and white matter on day 14. Carbachol significantly reduced the number of microglias in the hippocampus and white matter on day 10 and in the cortex on days 10 and 14. CONCLUSION: The main area of late inflammation was the cortex. Repetitive administration of carbachol reduces early and late inflammation after HI in the developing brain.

Antispasmodic effects of yarrow (Achillea millefolium L.) extract in the isolated ileum of rat.

Achillea millefolium L. is cultivated in Iran and widely used in traditional medicine for gastrointestinal disorders. The aim of this study was to determine the effect of hydroalcoholic extract of A. millefolium on the contraction and relaxation of isolated ileum in rat. In this experimental study, aerial parts of A. millefolium were extracted by maceration in ethanol 70% for 72 h. Terminal portion of ileum in 100 male Wistar rats was dissected and its contractions were recorded isotonically in an organ bath containing Tyrode solution (37 °C, pH 7.4) under one gram tension. Acetylcholine (1mM) and KCl (60mM) were used to create isotonic contractions. Propranolol and Nω-Nitro-L-arginine methylester hydrochloride (L-NAME) were used to investigate the mechanisms of action prior to giving the extract to the relevant groups. Data were compared by ANOVA and Turkey's post hoc test.. The results showed that the ileum contraction was induced by KCl and acetylcholine induced contraction was significantly reduced by A. millefolium extract. The cumulative concentrations of A. millefolium relaxed the KCl and acetylcholine induced contractions (n=14, p<0.001). The inhibitory effect of extract on contraction induced by KCl and acetylcholine was not significantly affected neither by propranolol (1µM) nor by L-NAME (100 µM). There was no significant difference in the rate of relaxation by propranolol and L-NAME between the two groups. In conclusion, A. millefolium can inhibit contraction of smooth muscle of ileum in rat, and it can be used for eliminating intestinal spasms. These results suggest that the relaxatory effect of A. millefolium on ileum contractions can be due to the blockade of voltage dependent calcium channels. In addition, the ß-adrenoceptors, cholinergic receptors and nitric oxide production are not powerful actors in inhibitory effect of A. millefolium. So, the nitric oxide and adrenergic systems may also be involved in the antispasmodic effect of A. millefolium.

Involvement of dopaminergic receptors of the rat nucleus accumbens in decreasing the conditioned place preference induced by lateral hypothalamus stimulation.

Our previous study showed that chemical stimulation of the lateral hypothalamus (LH) by carbachol can produce conditioned place preference (CPP) in rats. Also, it has been indicated that orexin activates the mesolimbic dopamine projecting neurons to the nucleus accumbens (NAc) and promotes the development of reward in rodents. Therefore, in this study, we tried to determine the role of intra-accumbal D1 and D2 dopamine receptors in the development (acquisition) of reward-related behaviors induced by chemical stimulation of the LH. Eighty-eight adult male Wistar rats were unilaterally implanted by two separate cannulae into the LH and NAc. For chemical stimulation of LH, carbachol (250nmol/0.5µl saline) was microinjected once daily during 3-days conditioning phase (acquisition period) of CPP paradigm. In the next experiments, different doses of D1 receptor antagonist, SCH23390 (0.25, 1 and 4µg/0.5µl saline) or sulpiride (0.25, 1 and 4µg/0.5µl DMSO) as a D2 receptor antagonist were unilaterally microinjected into the NAc, 5min prior to LH stimulation. One-way ANOVA showed that intra-accumbal administration of SCH23390 or sulpiride can decrease the development of LH stimulation-induced CPP in the rats. However, this decrease is more effective after blockade of the D2 dopamine receptor in the NAc. It seems that the dopaminergic system in this area is involved in place preference induced by LH stimulation.

Effect of arecoline on regeneration of injured peripheral nerves.

The present study provides in vitro and in vivo evaluation of arecoline on peripheral nerve regeneration. In the in vitro study, we found that arecoline at 50 µg/ml could significantly promote the survival and outgrowth of cultured Schwann cells as compared to the controls treated with culture medium only. In the in vivo study, we evaluated peripheral nerve regeneration across a 10-mm gap in the sciatic nerve of the rat, using a silicone rubber nerve chamber filled with the arecoline solution. In the control group, the chambers were filled with normal saline only. At the end of the fourth week, morphometric data revealed that the arecoline-treated group at 5 µg/ml significantly increased the number and the density of myelinated axons as compared to the controls. Immunohistochemical staining in the arecoline-treated animals at 5 µg/ml also showed their neural cells in the L4 and L5 dorsal root ganglia ipsilateral to the injury were strongly retrograde-labeled with fluorogold and lamina I-II regions in the dorsal horn ipsilateral to the injury were significantly calcitonin gene-related peptide-immunolabeled compared with the controls. In addition, we found that the number of macrophages recruited in the distal sciatic nerve was increased as the concentration of arecoline was increased. Electrophysiological measurements showed the arecoline-treated groups at 5 and 50 µg/ml had a relatively larger nerve conductive velocity of the evoked muscle action potentials compared to the controls. These results indicate that arecoline could stimulate local inflammatory conditions, improving the recovery of a severe peripheral nerve injury.