Clozapine is a second generation antipsychotic drug that has proven to be helpful in the management of patients with psychotic disorders that are resistant to other medications. Unfortunately, the majority of patients treated with clozapine develop metabolic dysregulation, including weight gain and insulin resistance. There are few treatments available to effectively counter these side-effects. The goal of the present study was to use an established animal model to better understand the nature of these metabolic side-effects and determine whether existing drugs could be used to alleviate metabolic changes. Adult female rats were treated with a range of doses of clozapine (2, 10 and 20 mg/kg) and subjected to the hyperinsulinemic-euglycemic clamp, to measure whole-body insulin resistance. Clozapine dose-dependently decreased the glucose infusion rate, reflecting pronounced insulin resistance. To reverse the insulin resistance, rats were co-treated with the ganglionic blocker mecamylamine (0.1, 1.0 and 5.0 mg/kg) which dose-dependently reversed the effects of 10 mg/kg clozapine. A 1.0 mg/kg dose of mecamylamine independently reversed the large increase in peripheral epinephrine caused by treatment with clozapine. To study the influence of specific adrenoceptors, rats were treated with multiple doses of α1 (prazosin), α2 (idazoxan), β1 (atenolol) and β2 (butoxamine) adrenoceptor antagonists after the onset of clozapine-induced insulin resistance. Both beta blockers were effective in attenuating the effects of clozapine, while idazoxan had a smaller effect; no change was seen with prazosin. The current results indicate that peripheral catecholamines may play a role in clozapine\'s metabolic effects and be a target for future treatments.

Ranunculus scleratus Linn. is used in folk medicine to treat hypertension. This study was aimed at providing validation to its traditional use and to explore underlying mechanisms of action. Effects of hydro-ethanolic crude extract of the plant and its fractions on blood pressure was evaluated using direct surgical method in normotensive and in fructose induced hypertensive rats. Various doses of crude extract, RSC, (5, 10, 20, 30mg/kg) and all fractions (3, 5, 10, 20mg/kg) were studied. Results suggested that aqueous fraction of R. scleratus (RSA) produced most pronounced effects at 10mg/kg in normotensive and at 20mg/kg in hypertensive animals. Underlying mechanisms, using various pharmacological antagonists were also elucidated. Results suggested the involvement of muscarinic receptor, angiotensin converting enzyme (ACE) inhibition, ganglionic block and nitric oxide (NO) release in presenting hypotensive response.

Previous studies showed that mecamylamine a noncompetitive and nonspecific blocker of nicotinic acetylcholine receptors (nAChRs), stimulates the activity of the dorsal raphe nucleus (DRN) serotonergic neurons and DRN serotonin (5-HT) release. In the present study, the mechanisms involved in these mecamylamine-induced effects were examined using electrophysiology and calcium-imaging studies, both performed in Wistar rat midbrain slices. Mecamylamine (0.5-9 μM), bath administered, increased the firing frequency of identified 5-HT DRN neurons by a maximum of 5% at 3 μM. This effect was accompanied by a 112 % increase in the frequency of spontaneous excitatory postsynaptic currents of 5-HT DRN neurons. It was blocked by the AMPA/kainate receptor blocker CNQX (10 μM) and by the specific α4β2 nAChRs blocker dihydro-β-erythroidine (100 nM) but was not affected by tetrodotoxin (TTX, 500 nM). Simultaneously, mecamylamine produced a 58 % decrease in the frequency of GABAergic spontaneous inhibitory postsynaptic currents, an effect that was not influenced by TTX. Calcium-imaging studies support the results obtained with the electrophysiological studies by showing that mecamylamine (3 μM) increases the activity of a cell population located in the midline of the DRN, which was sensitive to the inhibitory effects of 8-OH-DPAT, an agonist at 5-HT1A receptors. It is assumed that mecamylamine, in low concentrations, acts as an agonist of α4β2 nAChRs present on the glutamatergic DRN terminals, thus increasing intra-raphe glutamate release. This stimulatory effect is reinforced by the decrease in DRN GABA release, which is dependent on the mecamylamine-induced blockade of α7 nAChRs located on DRN GABAergic terminals. We hypothesize that at least a part of mecamylamine antidepressant effects described in animal models of depression are mediated by an increase in DRN 5-HT release.

The skin temperature (Tm) of the orofacial area influences orofacial functions and is related to the blood flow (BF). Marked increases in BF mediated by parasympathetic vasodilation may be important for orofacial Tm regulation. Therefore, we examined the relationship between parasympathetic reflex vasodilation and orofacial Tm in anesthetized rats. Electrical stimulation of the central cut end of the lingual nerve (LN) elicited significant increases in BF and Tm in the lower lip. These increases were significantly reduced by hexamethonium, but not atropine. VIP agonist increased both BF and Tm in the lower lip. The activation of the superior cervical sympathetic trunk (CST) decreased BF and Tm in the lower lip; however, these decreases were significantly inhibited by LN stimulation. Our results suggest that parasympathetic vasodilation plays an important role in the maintaining the hemodynamics and Tm in the orofacial area, and that VIP may be involved in this response.

The mechanisms underlying electrical rhythmicity in smooth muscle of the proximal colon are incompletely understood. Our aim was to identify patterns of electrical rhythmicity in smooth muscle of the proximal region of isolated whole mouse colon and characterize their mechanisms of origin. Two independent extracellular recording electrodes were used to record the patterns of electrical activity in smooth muscle of the proximal region of whole isolated mouse colon. Cross-correlation analysis was used to quantify spatial coordination of these electrical activities over increasing electrode separation distances. Four distinct neurogenic patterns of electrical rhythmicity were identified in smooth muscle of the proximal colon, three of which have not been identified and consisted of bursts of rhythmic action potentials at 1-2 Hz that were abolished by hexamethonium. These neurogenic patterns of electrical rhythmicity in smooth muscle were spatially and temporally synchronized over large separation distances (≥2 mm rosto-caudal axis). Myogenic slow waves could be recorded from the same preparations, but they showed poor spatial and temporal coordination over even short distances (≤1 mm rostro-caudal axis). It is not commonly thought that electrical rhythmicity in gastrointestinal smooth muscle is dependent upon the enteric nervous system. Here, we identified neurogenic patterns of electrical rhythmicity in smooth muscle of the proximal region of isolated mouse colon, which are dependent on synaptic transmission in the enteric nervous system. If the whole colon is studied in vitro, recordings can preserve novel neurogenic patterns of electrical rhythmicity in smooth muscle.NEW & NOTEWORTHY Previously, it has not often been thought that electrical rhythmicity in smooth muscle of the gastrointestinal tract is dependent upon the enteric nervous system. We identified patterns of electrical rhythmicity in smooth muscle of the mouse proximal colon that were abolished by hexamethonium and involved the temporal synchronization of smooth muscle membrane potential over large spatial fields. We reveal different patterns of electrical rhythmicity in colonic smooth muscle that are dependent on the ENS.

Cyclical propagating waves of muscle contraction have been recorded in isolated small intestine or colon, referred to here as motor complexes (MCs). Small intestinal and colonic MCs are neurogenic, occur at similar frequencies, and propagate orally or aborally. Whether they can be coordinated between the different gut regions is unclear. Motor behavior of whole length mouse intestines, from duodenum to terminal rectum, was recorded by intraluminal multisensor catheter. Small intestinal MCs were recorded in 27/30 preparations, and colonic MCs were recorded in all preparations (n = 30) with similar frequencies (0.54 ± 0.03 and 0.58 ± 0.02 counts/min, respectively). MCs propagated across the ileo-colonic junction in 10/30 preparations, forming \"full intestine\" MCs. The cholinesterase inhibitor physostigmine increased the probability of a full intestine MC but had no significant effect on frequency, speed, or direction. Nitric oxide synthesis blockade by Nω-nitro-l-arginine, after physostigmine, increased MC frequency in small intestine only. Hyoscine-resistant MCs were recorded in the colon but not small intestine (n = 5). All MCs were abolished by hexamethonium (n = 18) or tetrodotoxin (n = 2). The enteric neural mechanism required for motor complexes is present along the full length of both the small and large intestine. In some cases, colonic MCs can be initiated in the distal colon and propagate through the ileo-colonic junction, all the way to duodenum. In conclusion, the ileo-colonic junction provides functional neural continuity for propagating motor activity that originates in the small or large intestine.NEW & NOTEWORTHY Intraluminal manometric recordings revealed motor complexes can propagate antegradely or retrogradely across the ileo-colonic junction, spanning the entire small and large intestines. The fundamental enteric neural mechanism(s) underlying cyclic motor complexes exists throughout the length of the small and large intestine.

Obesity-related hypertension is a major public health concern. We recently demonstrated that plasma levels of the soluble form of the prorenin receptor (sPRR) were elevated in obesity-associated hypertension. Therefore, in the present study, we investigated the contribution of sPRR to blood pressure (BP) elevation in the context of obesity. High fat-fed C57BL/6 male mice were infused with vehicle or sPRR (30 µg/kg per day) via subcutaneously implanted osmotic minipump for 4 weeks. BP parameters were recorded using radiotelemetry devices. Male mice infused with sPRR exhibited higher systolic BP and mean arterial pressure and lower spontaneous baroreflex sensitivity than mice infused with vehicle. To define mechanisms involved in systolic BP elevation, mice were injected with an AT1R (Ang II [angiotensin II] type 1 receptor) antagonist (losartan), a muscarinic receptor antagonist (atropine), a β-adrenergic antagonist (propranolol), and a ganglionic blocker (chlorisondamine). Losartan did not blunt sPRR-induced elevation in systolic BP. Chlorisondamine treatment exacerbated the decrease in mean arterial pressure in male mice infused with sPRR. These results demonstrated that sPRR induced autonomic nervous dysfunction. Interestingly, plasma leptin levels were increased in high fat-fed C57BL/6 male mice infused with sPRR. Overall, our results indicated that sPRR increased systolic BP through an impairment of the baroreflex sensitivity and an increase in the sympathetic tone potentially mediated by leptin in high fat-fed C57BL/6 male mice.

What strategies are employed by the sympathetic system to communicate with the circulation? Muscle sympathetic nerve activity (MSNA) occurs in bursts of synchronous action potential (AP) discharge, yet whether between-burst asynchronous AP firing exists remains unknown. Using multiunit microneurography and a continuous wavelet transform to isolate APs, we studied AP synchronicity within human MSNA. Asynchronous APs were defined as those which occurred between bursts. Experiment 1 quantified AP synchronicity in eight individuals at baseline (BSL), -10 mmHg lower body negative pressure (LBNP), -40 mmHg LBNP, and end-expiratory apnea (APN). At BSL, 33 ± 12% of total AP activity was asynchronous. Asynchronous discharge was unchanged from BSL (67 ± 37 AP/min) to -10 mmHg LBNP (69 ± 33 AP/min), -40 mmHg LBNP (83 ± 68 AP/min), or APN (62 ± 39 AP/min). Across all conditions, asynchronous AP probability and frequency decreased with increasing AP size. Experiment 2 examined the impact of the ganglia on AP synchronicity by using nicotinic blockade (trimethaphan). The largest asynchronous APs were derecruited from BSL (11 ± 4 asynchronous AP clusters) to the last minute of the trimethaphan infusion with visible bursts (7 ± 2 asynchronous AP clusters). However, the 6 ± 2 smallest asynchronous AP clusters could not be blocked by trimethaphan and persisted to fire 100 ± 0% asynchronously without forming bursts. Nonnicotinic ganglionic mechanisms affect some, but not all, asynchronous activity. The fundamental behavior of human MSNA contains between-burst asynchronous AP discharge, which accounts for a considerable amount of BSL activity.NEW & NOTEWORTHY Historically, sympathetic nerve activity destined for the blood vessels supplying skeletal muscle (MSNA) has been characterized by spontaneous bursts formed by synchronous action potential (AP) discharge. However, this study found a considerable amount (~30% during baseline) of sympathetic AP discharge to fire asynchronously between bursts of human MSNA. Trimethaphan infusion revealed that nonnicotinic ganglionic mechanisms contribute to some, but not all, asynchronous discharge. Asynchronous sympathetic AP discharge represents a fundamental behavior of MSNA.

Our previous immunoprecipitation analysis of nicotinic acetylcholine receptors (nAChRs) in the mouse superior cervical ganglion (SCG) revealed that approximately 55%, 24%, and 21% of receptors are comprised of α3β4, α3β4α5, and α3β4β2 subunits, respectively. Moreover, mice lacking β4 subunits do not express α5-containing receptors but still express a small number of α3β2 receptors. Here, we investigated how synaptic transmission is affected in the SCG of α5β4-KO and α5β2-KO mice. Using an ex vivo SCG preparation, we stimulated the preganglionic cervical sympathetic trunk and measured compound action potentials (CAPs) in the postganglionic internal carotid nerve. We found that CAP amplitude was unaffected in α5β4-KO and α5β2-KO ganglia, whereas the stimulation threshold for eliciting CAPs was significantly higher in α5β4-KO ganglia. Moreover, intracellular recordings in SCG neurons revealed no difference in EPSP amplitude. We also found that the ganglionic blocking agent hexamethonium was the most potent in α5β4-KO ganglia (IC50 : 22.1 μmol/L), followed by α5β2-KO (IC50 : 126.7 μmol/L) and WT ganglia (IC50 : 389.2 μmol/L). Based on these data, we estimated an IC50 of 568.6 μmol/L for a receptor population consisting solely of α3β4α5 receptors; and we estimated that α3β4α5 receptors comprise 72% of nAChRs expressed in the mouse SCG. Similarly, by measuring the effects of hexamethonium on ACh-induced currents in cultured SCG neurons, we found that α3β4α5 receptors comprise 63% of nAChRs. Thus, in contrast to our results obtained using immunoprecipitation, these data indicate that the majority of receptors at the cell surface of SCG neurons consist of α3β4α5.

The role of the neurogenic pathway in early phases of cardioprotection during remote ischemic preconditioning (RIPC) and adenosine preconditioning is reported.
This study was designed to explore the involvement of the neurogenic pathway in late phases of cardioprotection during RIPC and adenosine preconditioning.
Fifty-four Wistar rats were used and divided into 9 experimental groups. RIPC was induced by tying the blood pressure cuff around the hind limb and subjecting to 4 cycles of inflation and deflation of 5 minutes each. In early RIPC, the heart was isolated immediately after the last episode of RIPC, whereas in late RIPC, the heart was isolated 24 hours after the last cycle of RIPC. In a similar way, adenosine preconditioning was instituted in early and late phases by either isolating the heart 40 minutes or 24 hours after adenosine (4 mg/kg, intraperitoneally [i.p.]) administration. Isolated hearts were subjected to ischemia-reperfusion (I/R) injury on the Langendorff\'s system.
Both early and late phases of RIPC and adenosine preconditioning significantly abrogated I/R-induced myocardial injury in terms of decrease in the release of lactate dehydrogenase, creatine kinase, and decrease in infarct size. Pretreatment with hexamethonium, a ganglion blocker (20 mg/kg, i.p.), significantly abolished the cardioprotective effects of both early and late phases of RIPC and adenosine preconditioning.
Apart from the involvement of the neurogenic pathway in the early phases, there is a critical role of the neurogenic pathway in the late phase of cardioprotection during RIPC and adenosine preconditioning.