Neuromodulation Leads to a Burst-Tonic Switch in a Subset of VIP Neurons in Mouse Primary Somatosensory (Barrel) Cortex.

Neocortical GABAergic interneurons expressing vasoactive intestinal polypeptide (VIP) contribute to sensory processing, sensorimotor integration, and behavioral control. In contrast to other major subpopulations of GABAergic interneurons, VIP neurons show a remarkable diversity. Studying morphological and electrophysiological properties of VIP cells, we found a peculiar group of neurons in layer II/III of mouse primary somatosensory (barrel) cortex, which showed a highly dynamic burst firing behavior at resting membrane potential that switched to tonic mode at depolarized membrane potentials. Furthermore, we demonstrate that burst firing depends on T-type calcium channels. The burst-tonic switch could be induced by acetylcholine (ACh) and serotonin. ACh mediated a depolarization via nicotinic receptors whereas serotonin evoked a biphasic depolarization via ionotropic and metabotropic receptors in 48% of the population and a purely monophasic depolarization via metabotropic receptors in the remaining cells. These data disclose an electrophysiologically defined subpopulation of VIP neurons that via neuromodulator-induced changes in firing behavior is likely to regulate the state of cortical circuits in a profound manner.

Attention-enhancing effects of propranolol and synergistic effects with nicotine.

Nicotine increases the output of every major neurotransmitter. In previous studies designed to identify the secondary neurotransmitter systems mediating nicotine's attention-enhancing effects in a rat model, the ß-adrenoceptor antagonist propranolol blocked these effects. The present study was designed to test whether this mechanism held true in humans, thus guiding development of novel nicotinic agonists for cognitive enhancement. Twenty-six nonsmokers completed a nicotine (7 mg/24 h transdermally) x propranolol (40 mg p.o., body weight-adjusted) interaction study. Over four test days, each participant received double-placebo, nicotine only, propranolol only, and nicotine plus propranolol in randomized sequence before cognitive testing. No drug effects were seen in a visuospatial attention task. In the Rapid Visual Information Processing Task, performed in two 15-min blocks, neither drug alone significantly affected hit rate, but both drugs combined acted synergistically to alleviate its decrement over time in the first block and displayed additive beneficial effects in the second. In a change detection task, propranolol enhanced accuracy and reduced reaction time independent of nicotine presence. Propranolol also enhanced subjective self-reports of vigor. Overall, the findings were contrary to those hypothesized. Propranolol displayed beneficial effects on cognition, especially on sustaining performance over time. ß-adrenoceptor activation by nicotine-induced noradrenaline release appeared to limit performance-enhancing effects of nicotine, because they were unmasked by ß-adrenoceptor antagonism. The results suggest that cognitive effects of changes in ß-adrenoceptor tone are context-dependent; contrary to rodent paradigms, human cognitive paradigms require no physical orienting in space but prolonged periods of remaining stationary while sustaining predictable processing demands.

Successful and unsuccessful attempts to swallow in a reduced Aplysia preparation regulate feeding responses and produce memory at different neural sites.

Sensory feedback shapes ongoing behavior and may produce learning and memory. Motor responses to edible or inedible food in a reduced Aplysia preparation were examined to test how sensory feedback affects behavior and memory. Feeding patterns were initiated by applying a cholinomimetic onto the cerebral ganglion. Feedback from buccal muscles increased the response variability and response rate. Repeated application of the cholinomimetic caused decreased responses, expressed in part by lengthening protractions. Swallowing strips of "edible" food, which in intact animals induces learning that enhances ingestion, increased the response rate, and shortened the protraction length, reflecting more swallowing. Testing memory by repeating the procedure prevented the decrease in response rate observed with the cholinomimetic alone, and shortened protractions. Training with "inedible" food that in intact animals produces learning expressed by decreased responses caused lengthened protractions. Testing memory by repeating the procedure did not cause decreased responses or lengthened protractions. After training and testing with edible or inedible food, all preparations were exposed to the cholinomimetic alone. Preparations previously trained with edible food displayed memory expressed as decreased protraction length. Preparations previously trained with inedible food showed decreases in many response parameters. Memory for inedible food may arise in part via a postsynaptic decrease in response to acetylcholine released by afferents sensing food. The lack of change in response number, and in the time that responses are maintained during the two training sessions preceding application of the cholinomimetic alone suggests that memory expression may differ from behavioral changes during training.

Neuromodulation-dependent effect of gated high-frequency, LFMS-like electric field stimulation in mouse cortical slices.

Low-field magnetic stimulation (LFMS) is a gated high-frequency non-invasive brain stimulation method (500 Hz gated at 2 Hz) with a proposed antidepressant effect. However, it has remained unknown how such stimulation paradigms modulate neuronal network activity and how the induced changes depend on network state. Here we examined the immediate and outlasting effects of the gated high-frequency electric field associated with LFMS on the cortical activity as a function of neuromodulatory tone that defines network state. We used a sham-controlled study design to investigate effects of stimulation (20 min of 0.5 s trains of 500 Hz charge-balanced pulse stimulation patterned at 0.5 Hz) on neural activity in mouse medial prefrontal cortex in vitro. Bath application of cholinergic and noradrenergic agents enabled us to examine the stimulation effects as a function of neuromodulatory tone. The stimulation attenuated the increase in firing rate of layer V cortical neurons during the post-stimulation period in the presence of cholinergic activation. The same stimulation had no significant immediate or outlasting effect in the absence of exogenous neuromodulators or in the presence of noradrenergic activation. These results provide electrophysiological insights into the neuromodulatory-dependent effects of gated high-frequency stimulation. More broadly, our results are the first to provide a mechanistic demonstration of how behavioral states and arousal levels may modify the effects of non-invasive brain stimulation.

Nicotinic α7 receptors enhance NMDA cognitive circuits in dorsolateral prefrontal cortex.

The cognitive function of the highly evolved dorsolateral prefrontal cortex (dlPFC) is greatly influenced by arousal state, and is gravely afflicted in disorders such as schizophrenia, where there are genetic insults in α7 nicotinic acetylcholine receptors (α7-nAChRs). A recent behavioral study indicates that ACh depletion from dlPFC markedly impairs working memory [Croxson PL, Kyriazis DA, Baxter MG (2011) Nat Neurosci 14(12):1510-1512]; however, little is known about how α7-nAChRs influence dlPFC cognitive circuits. Goldman-Rakic [Goldman-Rakic (1995) Neuron 14(3):477-485] discovered the circuit basis for working memory, whereby dlPFC pyramidal cells excite each other through glutamatergic NMDA receptor synapses to generate persistent network firing in the absence of sensory stimulation. Here we explore α7-nAChR localization and actions in primate dlPFC and find that they are enriched in glutamate network synapses, where they are essential for dlPFC persistent firing, with permissive effects on NMDA receptor actions. Blockade of α7-nAChRs markedly reduced, whereas low-dose stimulation selectively enhanced, neuronal representations of visual space. These findings in dlPFC contrast with the primary visual cortex, where nAChR blockade had no effect on neuronal firing [Herrero JL, et al. (2008) Nature 454(7208):1110-1114]. We additionally show that α7-nAChR stimulation is needed for NMDA actions, suggesting that it is key for the engagement of dlPFC circuits. As ACh is released in cortex during waking but not during deep sleep, these findings may explain how ACh shapes differing mental states during wakefulness vs. sleep. The results also explain why genetic insults to α7-nAChR would profoundly disrupt cognitive experience in patients with schizophrenia.

M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity.

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments.

Fractional flow reserve with dobutamine challenge and coronary microvascular endothelial dysfunction in symptomatic myocardial bridging.

BACKGROUND: Myocardial bridging (MB) results in compression of the coronary artery lumen in systole, extending into diastole with resultant hemodynamic alternation as reflected by fractional flow reserve (FFR). MB has also been associated with coronary endothelial dysfunction. The objective of this study was to investigate relationship between FFR with dobutamine challenge and coronary microvascular endothelial dysfunction in symptomatic MB. METHODS AND RESULTS: Seventeen consecutive patients who had cardiac catheterization assessment of MB were enrolled. The patients were divided into 2 groups according to normal (% increase in coronary blood flow [%CBF] ≥50%, n=7) or impaired (%CBF <50%, n=10) coronary microvascular endothelial function assessed on vasoreactivity in the coronary artery with intracoronary infusion of acetylcholine (Ach). Myocardial ischemia was then assessed using FFR at rest and during i.v. dobutamine infusion challenge across the MB with intracoronary pressure wires. FFR was significantly decreased at peak dobutamine infusion compared to at rest in the impaired group (0.85±0.06 vs. 0.91±0.05, P=0.001), but not in the normal group (0.93±0.05 vs. 0.91±0.07, P=0.618). Both FFR at rest and at peak dobutamine infusion had a positive correlation with %CBF by Ach in the impaired group (r(2)=0.46, P=0.030; r(2)=0.52, P=0.018, respectively). CONCLUSIONS: Microvascular endothelial dysfunction was associated with decreased FFR at peak dobutamine stress in patients with symptomatic MB.

Striatal origin of the pathologic beta oscillations in Parkinson's disease.

Enhanced oscillations at beta frequencies (8-30 Hz) are a signature neural dynamic pathology in the basal ganglia and cortex of Parkinson's disease patients. The mechanisms underlying these pathological beta oscillations remain elusive. Here, using mathematical models, we find that robust beta oscillations can emerge from inhibitory interactions between striatal medium spiny neurons. The interaction of the synaptic GABAa currents and the intrinsic membrane M-current promotes population oscillations in the beta frequency range. Increased levels of cholinergic drive, a condition relevant to the parkinsonian striatum, lead to enhanced beta oscillations in the striatal model. We show experimentally that direct infusion of the cholinergic agonist carbachol into the striatum, but not into the neighboring cortex, of the awake, normal rodent induces prominent beta frequency oscillations in the local field potential. These results provide evidence for amplification of normal striatal network dynamics as a mechanism responsible for the enhanced beta frequency oscillations in Parkinson's disease.

Targeting α-7 nicotinic acetylcholine receptor in the enteric nervous system: a cholinergic agonist prevents gut barrier failure after severe burn injury.

We have previously shown that vagal nerve stimulation prevents intestinal barrier loss in a model of severe burn injury in which injury was associated with decreased expression and altered localization of intestinal tight junction proteins. α-7 Nicotinic acetylcholine receptor (α-7 nAchR) has been shown to be necessary for the vagus nerve to modulate the systemic inflammatory response, but the role of α-7 nAchR in mediating gut protection remained unknown. We hypothesized that α-7 nAchR would be present in the gastrointestinal tract and that treatment with a pharmacological agonist of α-7 nAchR would protect against burn-induced gut barrier injury. The effects of a pharmacological cholinergic agonist on gut barrier integrity were studied using an intraperitoneal injection of nicotine 30 minutes after injury. Intestinal barrier integrity was examined by measuring permeability to 4-kDa fluorescein isothiocyanate-dextran and by examining changes in expression and localization of the intestinal tight junction proteins occludin and ZO-1. Nicotine injection after injury prevented burn-induced intestinal permeability and limited histological gut injury. Treatment with nicotine prevented decreased expression and altered localization of occludin and ZO-1, as seen in animals undergoing burn alone. Defining the interactions among the vagus nerve, the enteric nervous system, and the intestinal epithelium may lead to development of targeted therapeutics aimed at reducing gut barrier failure and intestinal inflammation after severe injury.

The expression of O(6) -methylguanine-DNA methyltransferase in human oral keratinocytes stimulated with arecoline.

BACKGROUND: O(6) -methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that can protect cells from carcinogenic effects of alkylating agents by removing adducts from the O(6) position of guanine. Evidences indicated that areca quid chewing may increase the risk of oral squamous cell carcinoma (OSCC). This study was to investigate the role of MGMT expression in OSCCs and the normal oral tissues. METHODS: Thirty-two OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by the immunohistochemistry for MGMT. Primary human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot. Nicotine, an important component of cigarette smoke, was added to find the possible regulatory mechanisms. RESULTS: Significant association was observed between low MGMT expression and advanced clinical stage of OSCCs and lymph node metastasis (P = 0.03). MGMT expression was significantly higher in patients only chewing areca quid than patients both chewing areca quid and smoking (P = 0.028). Arecoline was found to elevate MGMT expression in a dose- and time-dependent manner. The addition of nicotine was found to enhance arecoline-induced MGMT expression. CONCLUSION: Our results indicate that MGMT could be used clinically as a predictive marker for tumor processing, the potential for lymph node metastasis as well as advanced clinical stage. MGMT expression was significantly upregulated by arecoline in HOKs. Nicotine has a synergistic effect of arecoline-induced MGMT expression. The cigarette smoking may act synergistically in the pathogenesis of OSCC in areca quid chewers via the upregulation of MGMT.

Intratympanic treatment for tinnitus: a review.

Since the 1940s, various attempts have been made to treat peripheral tinnitus by way of intratympanic injection. This administration procedure requires only low concentrations of medication, thanks to the highly targeted delivery to the site of action and comes with minimal systemic exposure. While different compounds have been tested for their effects on tinnitus by intratympanic injection, there has been no breakthrough so far. Accordingly, the clinical use of intratympanic tinnitus treatments has remained limited to date. A more widespread adoption of this approach will require the development of specific medications for peripheral tinnitus, as well as proof of safety and efficacy, which would be determined from randomized controlled clinical trials.

Unprovoked atrial tachyarrhythmias in aging spontaneously hypertensive rats: the role of the autonomic nervous system.

Experimental models of unprovoked atrial tachyarrhythmias (AT) in conscious, ambulatory animals are lacking. We hypothesized that the aging, spontaneously hypertensive rat (SHR) may provide such a model. Baseline ECG recordings were acquired with radiotelemetry in eight young (14-wk-old) and eight aging (55-wk-old) SHRs and in two groups of four age-matched Wistar-Kyoto (WKY) rats. Quantification of AT and heart rate variability (HRV) analysis were performed based on 24-h ECG recordings in unrestrained rats. All animals were submitted to an emotional stress protocol (air-jet). In SHRs, carbamylcholine injections were also performed. Spontaneous AT episodes were observed in all eight aging SHRs (median, 91.5; range, 4-444 episodes/24 h), but not in young SHRs or WKY rats. HRV analysis demonstrated significantly decreased low frequency components in aging SHRs compared with age-matched WKY rats (P < 0.01) and decreased low/high frequency ratios in both young (P < 0.01) and aging (P = 0.01) SHRs compared with normotensive controls. In aging SHRs, emotional stress significantly reduced the number of arrhythmic events, whereas carbamylcholine triggered AT and significantly increased atrial electrical instability. This study reports the occurrence of unprovoked episodes of atrial arrhythmia in hypertensive rats, and their increased incidence with aging. Our results suggest that autonomic imbalance with relative vagal hyperactivity may be responsible for the increased atrial arrhythmogenicity observed in this model. We also provide evidence that, in this model, the sympatho-vagal imbalance preceded the occurrence of arrhythmia. These results indicate that aging SHRs may provide valuable insight into the understanding of atrial arrhythmias.

Sleep-disordered breathing is an independent risk factor of aborted sudden cardiac arrest in patients with coronary artery spasm.

BACKGROUND: Sleep-disordered breathing (SDB) is often associated with sudden cardiac arrest (SCA) during sleep. Coronary artery spasm (CS) also occurs during sleep and is rarely associated with SCA, but the role of SDB in the risk of SCA is unknown in CS patients. This study evaluated the breathing patterns during sleep in CS patients with a prior history of aborted SCA. METHODS AND RESULTS: This study enrolled 24 patients (age 61.6 ± 11.0 years, male/female 19/5) with CS proven by an acetylcholine provocation test. They were divided into 2 groups: prior history of aborted SCA due to fatal arrhythmia (SCA group; n=9) and no such history (no-SCA group; n=15). Patients underwent overnight polysomnography with ambulatory electrocardiography. The overall prevalence of SDB (apnea hypopnea index ≥15) was 45.8% in this cohort. SDB was more frequent in the SCA group than in the no-SCA group (88.9% vs. 20.0% P=0.001) and identified as a pivotal risk factor of aborted SCA (odds ratio: 38.9, 95% CI: 2.80-1,498.2, P=0.01). Very-low-frequency was significantly correlated with the apnea hypopnea index in patients with SCA (P=0.01, r=0.78) during sleep. CONCLUSIONS: SDB is a significant risk factor for SCA in CS patients and autonomic instability during sleep might be involved in this association.

A secretagogue-small interfering RNA conjugate confers resistance to cytotoxicity in a cell model of Sjögren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS) is characterized by xerophthalmia and xerostomia resulting from loss of secretory function due to immune cell infiltration in lacrimal and salivary glands. Current therapeutic strategies for SS use secretagogues to induce secretion via muscarinic receptor stimulation. The purpose of this study was to create a secretagogue-small interfering RNA (siRNA) conjugate to deliver siRNA into cells via receptor-mediated endocytosis, thereby altering epithelial cell responses to external cues, such as proinflammatory or death signals, while simultaneously stimulating secretion. METHODS: Based on our expertise with type 3 muscarinic receptor (M3), we used carbachol, a ligand specific for muscarinic receptor, as the secretagogue. Carbachol was synthesized with an active choline group and was conjugated with an siRNA that targets caspase 3. A human salivary gland (HSG) cell line was used to test the efficacy of this secretagogue-siRNA conjugate. RESULTS: Lipofectamine transfection of the conjugate into HSG cells resulted in a 78% reduction in the expression of the caspase 3 gene, while external conjugate treatment of HSG cells resulted in intracellular calcium release and induction of endocytosis at levels similar to those of carbachol stimulation, indicating that the siRNA and carbachol portions of the conjugate retained their function after conjugation. HSG cells treated with conjugate (without Lipofectamine transfection) exhibited a 50% reduction in caspase 3 gene and protein expression, indicating that our conjugate was effective in delivering functional siRNA into cells via receptor-mediated endocytosis. Furthermore, tumor necrosis factor α-induced apoptosis was significantly reduced in conjugate-treated cells. CONCLUSION: Our secretagogue-siRNA conjugate prevented cytokine-induced apoptosis in salivary gland epithelial cells, which is critical to maintaining fluid secretion and potentially reversing the clinical hallmark of SS.

Effect of intracameral carbachol given during cataract surgery on macular thickness.

To evaluate the effect of intracameral carbachol on foveal thickness in patients who underwent uneventful cataract surgery. This retrospective study included two groups: the study group patients (group 1, n = 47 eyes) had uneventful cataract surgery and received only carbachol 0.01 % for miosis; the control group patients (group 2, n = 49 eyes) had uneventful cataract surgery without carbachol or any intracameral medication(s). The groups were compared for foveal thickness after cataract surgery. All phacoemulsification plus intraocular lens implantation surgeries were performed under local anesthesia via temporal clear corneal tunnel incisions. Mean values and standard deviations were calculated for preoperative and postoperative visual acuity (VA) and foveal thickness (FT) at 1 and 4 weeks. Optical coherence tomography was used for the FT measurements, with the MM6 map program. The patients in the study and control groups had a mean age of 57.78 ± 9.07 and 59.72 ± 8.96, respectively (p = 0.355). All eyes had a significant improvement in VA. In the study group, the mean FT at the visits before and 1 and 4 weeks after surgery was 216.87 ± 21.06, 228.81 ± 30.52, and 222.94 ± 29.91 µm, respectively. For the control group, the mean FT, before and 1 and 4 weeks after surgery, was 222.53 ± 17.66, 231.67 ± 23.08, and 225.41 ± 22.59 µm, respectively. Intracameral carbachol 0.01 % had no effect on foveal thickness in patients who underwent uneventful cataract surgery.

Cholinergic neural signals to the spleen down-regulate leukocyte trafficking via CD11b.

The cholinergic anti-inflammatory pathway is a physiological mechanism that inhibits cytokine production and diminishes tissue injury during inflammation. Recent studies demonstrate that cholinergic signaling reduces adhesion molecule expression and chemokine production by endothelial cells and suppresses leukocyte migration during inflammation. It is unclear how vagus nerve stimulation regulates leukocyte trafficking because the vagus nerve does not innervate endothelial cells. Using mouse models of leukocyte trafficking, we show that the spleen, which is a major point of control for cholinergic modulation of cytokine production, is essential for vagus nerve-mediated regulation of neutrophil activation and migration. Administration of nicotine, a pharmacologic agonist of the cholinergic anti-inflammatory pathway, significantly reduces levels of CD11b, a beta(2)-integrin involved in cell adhesion and leukocyte chemotaxis, on the surface of neutrophils in a dose-dependent manner and this function requires the spleen. Similarly, vagus nerve stimulation significantly attenuates neutrophil surface CD11b levels only in the presence of an intact and innervated spleen. Further mechanistic studies reveal that nicotine suppresses F-actin polymerization, the rate-limiting step for CD11b surface expression. These studies demonstrate that modulation of leukocyte trafficking via cholinergic signaling to the spleen is a specific, centralized neural pathway positioned to suppress the excessive accumulation of neutrophils at inflammatory sites. Activating this mechanism may have important therapeutic potential for preventing tissue injury during inflammation.

Carbachol alleviates rat cytokine release and organ dysfunction induced by lipopolysaccharide.

BACKGROUND: To observe the influence of carbachol on inflammatory cytokine release and its protective role on organ function in rat endotoxemia model, and, furthermore, to investigate its receptor mechanism in rat peritoneal macrophages in vitro. METHODS: In the animal experiments, Wistar rats were subjected to lipopolysaccharide (LPS) injection (5 mg/kg body weight) to establish an endotoxemia animal model, and carbachol/nicotine was given 15 minutes after LPS injection. Serum contents of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were determined with enzyme-linked immunosorbent assay 4 hours after LPS injection. Plasma alanine aminotransferase, creatine kinase-MB, and diamine oxidase contents were detected 24 hours after LPS injection. In cell experiments, rat peritoneal macrophages were collected and initially pretreated with atropine (muscarinic cholinergic receptor antagonist) or α-Bungarotoxin (an antagonist that specifically binds α7 subunit of nicotinic cholinergic receptor), then with carbachol or nicotine, and finally stimulated with LPS. Contents of TNF-α, IL-6, and IL-10 in supernatant were assayed by enzyme-linked immunosorbent assay. Furthermore, macrophages were exposed to nicotine and carbachol of high concentration and then stained with fluorescein isothiocyanate-labeled α-bungarotoxin and observed with fluorescent confocal microscopy. RESULTS: Carbachol inhibited expression of TNF-α and IL-6 after LPS injection and had no significant effect on IL-10 in rat endotoxemia model. It also inhibited the increase of plasma alanine aminotransferase and creatine kinase-MB contents whereas restored the inhibited plasma diamine oxidase activity. Cell experiments also showed that increases of TNF-α and IL-6 after LPS stimulation could be significantly inhibited by carbachol or nicotine, whereas IL-10 was not apparently altered. Atropine did not downregulate the inhibitive effects of both carbachol and nicotine, whereas α-bungarotoxin significantly downregulated these effects. Fluorescent confocal microscopy showed that nicotine and carbachol pretreatment markedly reduced the intensity of binding between fluorescein isothiocyanate-labeled α-bungarotoxin and macrophages. CONCLUSION: The results suggested that both carbachol and nicotine play a role in the anti-inflammatory process and organ function protection through the α7 subunit of nicotinic cholinergic receptor.

CD31+ T cells, endothelial function and cardiovascular risk.

Deficits in endothelial cell repair mechanisms are thought to contribute to the aetiology of endothelial dysfunction and, subsequently, cardiovascular disease (CVD). CD31(+) T cells or so-called "angiogenic T cells" are a newly defined T cell subset that exhibit favourable vascular qualities and show a strong negative relation with atherosclerotic disease severity. Despite growing evidence that CD31(+) T cells are important for vascular homeostasis, it is currently unknown if CD31(+) T cell number and function are related to endothelial function and CVD risk in healthy adults. To address this question, we studied 24 healthy adult men (ages: 21-70). Endothelial function was assessed by the forearm blood flow (FBF) response to intra-arterial infusion of acetylcholine (ACh) and CVD risk was estimated by Framingham Risk Score (FRS). CD31(+) T cell number was determined by fluorescence-activated cell sorting. Magnetic-activated cell sorting was used to isolate CD31(+) T cells for Boyden chamber migration. No relation was observed between CD31(+) T cell number and FBF response to ACh or FRS. However, CD31(+) T cell migration to stromal cell-derived factor (SDF)-1α and vascular endothelial growth factor (VEGF) was positively correlated with FBF response to ACh (r = 0.43 for SDF-1α; r = 0.38 for VEGF; both P<0.05) and inversely related to FRS (r = -0.53 for SDF-1α; r = -0.48 for VEGF; both P<0.05). These findings demonstrate that CD31(+) T cell function, but not number, is associated with in vivo endothelial function and CVD risk in healthy adult men.

Carbachol induces headache, but not migraine-like attacks, in patients with migraine without aura.

Carbachol induces headache in healthy subjects, but the migraine eliciting effect of carbachol has not previously been studied. We hypothesized that the cholinomimetic agonist carbachol would induce headache and migraine-like attacks in migraineurs. Carbachol (3 µg/kg) or placebo was randomly infused into 18 patients with migraine without aura in a double-blind crossover study. Headache was scored on a verbal rating scale from 0 to 10. Velocity in the middle cerebral artery (V(MCA)) and diameter of the superficial temporal artery (STA) were recorded. Fifteen patients experienced headache after carbachol compared with eight after placebo (P = 0.039). There was no difference in incidence of migraine-like attacks after carbachol (n = 8) compared with placebo (n = 6) (P = 0.687). Carbachol caused a decrease in V(MCA) (P = 0.044), but no change in STA (P = 0.089) compared with placebo. The study demonstrated that carbachol provocation is not a good model for experimental migraine.