Imaging in vivo acetylcholine release in the peripheral nervous system with a fluorescent nanosensor.

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological diseases. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and α-bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 to 358 µM. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose dependent, reversible, and we observe a reduction of ∼76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.

TRPM8: A Therapeutic Target for Neuroinflammatory Symptoms Induced by Severe Dry Eye Disease.

Dry eye disease (DED) is commonly associated with ocular surface inflammation and pain. In this study, we evaluated the effectiveness of repeated instillations of transient receptor potential melastatin 8 (TRPM8) ion channel antagonist M8-B on a mouse model of severe DED induced by the excision of extra-orbital lacrimal and Harderian glands. M8-B was topically administered twice a day from day 7 until day 21 after surgery. Cold and mechanical corneal sensitivities and spontaneous ocular pain were monitored at day 21. Ongoing and cold-evoked ciliary nerve activities were next evaluated by electrophysiological multi-unit extracellular recording. Corneal inflammation and expression of genes related to neuropathic pain and inflammation were assessed in the trigeminal ganglion. We found that DED mice developed a cold allodynia consistent with higher TRPM8 mRNA expression in the trigeminal ganglion (TG). Chronic M8-B instillations markedly reversed both the corneal mechanical allodynia and spontaneous ocular pain commonly associated with persistent DED. M8-B instillations also diminished the sustained spontaneous and cold-evoked ciliary nerve activities observed in DED mice as well as inflammation in the cornea and TG. Overall, our study provides new insight into the effectiveness of TRPM8 blockade for alleviating corneal pain syndrome associated with severe DED, opening a new avenue for ocular pain management.

Lidocaine Versus Bupivacaine in the Treatment of Headache with Intranasal Sphenopalatine Nerve Block.

BACKGROUND: Intranasal sphenopalatine ganglion (SPG) block has been shown to be an effective treatment for headaches. Multiple therapeutic agents have been studied, although the wide availability and low cost of lidocaine and bupivacaine have made them attractive treatment options. To the authors knowledge, no study has yet demonstrated superiority of one anesthetic over the other. OBJECTIVE: To determine the efficacy of lidocaine versus bupivacaine when performing intranasal sphenopalatine ganglion (SPG) block for the treatment of headaches. STUDY DESIGN: Retrospective cohort study. SETTING: A single tertiary care academic institutionMETHODS: This retrospective study identified patients who underwent SPG block at a single institution from January 1, 2014 to December 20, 2017. Patients were included if they were treated with either lidocaine or bupivacaine and had both pre- and post-procedure pain scores recorded on a 0-10 scale. Patients were excluded if they were less than 18 years of age. RESULTS: 386 total procedures were performed. 303 (78.5%) were lidocaine delivered via the SphenoCath device, and 83 (21.5%) were bupivacaine delivered via the Tx360 device. 90.2% of treatments (n = 348) decreased the patient's pain level. Of the treatments performed with lidocaine, 89.1% (n = 270) resulted in improvement of the patient's pain level with a mean decrease in pain level of 3.1 (SD ± 2.3). Of the treatments performed with bupivacaine, 94.0% (n = 78) resulted in improvement of the patient's pain level, with a mean decrease in pain level of 3.0 (SD ± 1.9). No statistically significant difference was found between the 2 anesthetics. LIMITATIONS: The retrospective study design may introduce selection bias. Both lidocaine and bupivacaine were administered by different devices (Sphenocath and Tx360 respectively) which may account for differences in initial treatment success. There were differences in the size of the two groups, which may also introduce error. CONCLUSIONS: This study demonstrates similar efficacy of SPG block performed with lidocaine or bupivacaine. While no difference was found, the particular advantages and disadvantages of the intranasal delivery device may influence physician choice. KEY WORDS: Sphenopalatine ganglion nerve block, lidocaine, bupivacaine, sphenocath, Tx360, pain intervetnio, headache, miimally invasive therapy.

Open-Label, Multi-Dose, Pilot Safety Study of Injection of OnabotulinumtoxinA Toward the Otic Ganglion for the Treatment of Intractable Chronic Cluster Headache.

BACKGROUND: The otic ganglion (OG) provides parasympathetic innervation to the cerebral circulation and cranial structures and may be involved in the pathophysiology of trigeminal autonomic headaches. This structure has never been targeted in any headache disorder. OBJECTIVE: To investigate the safety of injecting onabotulinumtoxin A (BTA) toward the OG in 10 patients with intractable chronic cluster headache and to collect efficacy data. METHODS: A total of 10 patients with chronic cluster headache were enrolled in this open-label, multi-dose pilot safety study. All patients were recruited and treated on an out-patient basis at St Olav's University Hospital (Norway). In 5 patients each, the OG was the injection target with 12.5 IU of BTA or 25 IU, respectively. The primary outcome measure was adverse events (AEs) and the main secondary outcome was the number of attacks per week measured at baseline and in the second month following injection. RESULTS: For the primary endpoint, we analyzed data for all 10 patients. There were a total of 17 AEs in 6 of the 10 patients. All AEs were considered mild and disappeared by the end of follow-up. The median number of attacks per week at baseline was 17.0 [7.8 to 25.8] vs 14.0 [7.3 to 20.0] in the second month following injection; difference: 3 (95%CI: -0.3 to 7.9), P = .063. CONCLUSIONS: Injection with BTA toward the OG appears to be safe. We did not find a statistically significant reduction in the number of attacks per week at month 2 after injection compared to the baseline. This study suggests that the OG is not an important target for the treatment of chronic cluster headache. A future study employing more precise targeting of the OG may be indicated.

Restoration of Cyclo-Gly-Pro-induced salivary hyposecretion and submandibular composition by naloxone in mice.

Cyclo-Gly-Pro (CGP) attenuates nociception, however its effects on salivary glands remain unclear. In this study, we investigated the acute effects of CGP on salivary flow and composition, and on the submandibular gland composition, compared with morphine. Besides, we characterized the effects of naloxone (a non-selective opioid receptor antagonist) on CGP- and morphine-induced salivary and glandular alterations in mice. After that, in silico analyses were performed to predict the interaction between CGP and opioid receptors. Morphine and CGP significantly reduced salivary flow and total protein concentration of saliva and naloxone restored them to the physiological levels. Morphine and CGP also reduced several infrared vibrational modes (Amide I, 1687-1594cm-1; Amide II, 1594-1494cm-1; CH2/CH3, 1488-1433cm-1; C = O, 1432-1365cm-1; PO2 asymmetric, 1290-1185cm-1; PO2 symmetric, 1135-999cm-1) and naloxone reverted these alterations. The in silico docking analysis demonstrated the interaction of polar contacts between the CGP and opioid receptor Cys219 residue. Altogether, we showed that salivary hypofunction and glandular changes elicited by CGP may occur through opioid receptor suggesting that the blockage of opioid receptors in superior cervical and submandibular ganglions may be a possible strategy to restore salivary secretion while maintaining antinociceptive action due its effects on the central nervous system.

Effect of pterygopalatine blockade on perioperative stress and inflammatory outcomes following paediatric cataract surgery.

BACKGROUND: General anesthesia is required to perform pediatric cataract surgery. To reduce severity of surgical intervention and postoperative complications, regional techniques have been concomitantly used. The traditional regional ophthalmic techniques are retrobulbar, peribulbar and sub-Tenon blocks, which present some technical difficulties and associated complication risks. The pterygopalatine blockade has been exempt of many of these concerns as it is performed out of the orbit. The purpose of this study was to compare the analgesic and anti-inflammatory effects of the pterygopalatine blockade with retrobulbar block in children undergoing elective congenital cataract surgery. METHODS: After approval of ethics committee and informed consents, patients were enrolled to the study to have either ultrasound-guided pterygopalatine block (group P) or retrobulbar block (group R), with 2 mL lidocaine 2% and 1 mL ropivacaine 0.5%. Hemodynamic monitoring was recorded throughout the perioperative period. Cortisol level and oxidation-reduction status were assessed before and after surgery. Pain and inflammatory response (Tyndall effect, corneal syndrome and edema) were assessed on the first postoperative day. RESULTS: Comparative analysis demonstrated a decrease in cortisol of 123.24% (p˂0.05) and an increase in the redox coefficient of 37.7% (p˂0.05) in group P. Pain intensity was significantly higher in group R until the 16th postoperative hour. The corneal syndrome in patients in group P and group R was noted by 7.6% and in 32.1%, respectively (p˂0.05). CONCLUSION: The use of the pterygopalatine blockade as a component of anesthesia in pediatric cataract surgery allows reduction of the severity of surgical stress during surgical intervention, providing intraoperative hemodynamic stability and prolonged analgesia.

Ketamine inhibits synaptic transmission and nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglia in situ.

The intravenous anaesthetic ketamine, has been demonstrated to inhibit nicotinic acetylcholine receptor (nAChR)-mediated currents in dissociated rat intracardiac ganglion (ICG) neurons (Weber et al., 2005). This effect would be predicted to depress synaptic transmission in the ICG and would account for the inhibitory action of ketamine on vagal transmission to the heart (Inoue and König, 1988). This investigation was designed to examine the activity of ketamine on (i) postsynaptic responses to vagal nerve stimulation, (ii) the membrane potential, and (iii) membrane current responses evoked by exogenous application of ACh and nicotine in ICG neurons in situ. Intracellular recordings were made using sharp intracellular microelectrodes in a whole mount ICG preparation. Preganglionic nerve stimulation and recordings in current- and voltage-clamp modes were used to assess the action of ketamine on ganglionic transmission and nAChR-mediated responses. Ketamine attenuated the postsynaptic responses evoked by nerve stimulation. This reduction was significant at clinically relevant concentrations at high frequencies. The excitatory membrane potential and current responses to focal application of ACh and nicotine were inhibited in a concentration-dependent manner by ketamine. In contrast, ketamine had no effect on either the directly-evoked action potential or excitatory responses evoked by focal application of γ-aminobutyric acid (GABA). Taken together, ketamine inhibits synaptic transmission and nicotine- and ACh-evoked currents in adult rat ICG. Ketamine inhibition of synaptic transmission and nAChR-mediated responses in the ICG contributes significantly to its attenuation of the bradycardia observed in response to vagal stimulation in the mammalian heart.

Control of Neurotransmission by NaV1.7 in Human, Guinea Pig, and Mouse Airway Parasympathetic Nerves.

Little is known about the neuronal voltage-gated sodium channels (NaVs) that control neurotransmission in the parasympathetic nervous system. We evaluated the expression of the α subunits of each of the nine NaVs in human, guinea pig, and mouse airway parasympathetic ganglia. We combined this information with a pharmacological analysis of selective NaV blockers on parasympathetic contractions of isolated airway smooth muscle. As would be expected from previous studies, tetrodotoxin potently blocked the parasympathetic responses in the airways of each species. Gene expression analysis showed that that NaV 1.7 was virtually the only tetrodotoxin-sensitive NaV1 gene expressed in guinea pig and human airway parasympathetic ganglia, where mouse ganglia expressed NaV1.1, 1.3, and 1.7. Using selective pharmacological blockers supported the gene expression results, showing that blocking NaV1.7 alone can abolish the responses in guinea pig and human bronchi, but not in mouse airways. To block the responses in mouse airways requires that NaV1.7 along with NaV1.1 and/or NaV1.3 is blocked. These results may suggest novel indications for NaV1.7-blocking drugs, in which there is an overactive parasympathetic drive, such as in asthma. The data also raise the potential concern of antiparasympathetic side effects for systemic NaV1.7 blockers.

Pilot study of sphenopalatine injection of onabotulinumtoxinA for the treatment of intractable chronic migraine.

Objective The main objective of this pilot study was to investigate the safety of administering onabotulinumtoxinA towards the sphenopalatine ganglion in 10 patients with intractable chronic migraine with an open, uncontrolled design. We also collected efficacy data to provide an indication as to whether future placebo-controlled studies should be performed. Method In a prospective, open-label, uncontrolled study after one-month baseline, we performed bilateral injections of 25 IU onabotulinumtoxinA (total dose 50 IU) toward the sphenopalatine ganglion in a single outpatient session in 10 patients with intractable migraine with a follow-up of 12 weeks. The primary outcome was adverse events and the main efficacy outcome was frequency of moderate and severe headache days in month 2 post-treatment compared to baseline. Results All 10 patients experienced a total of 25 adverse events. The majority of these were different types of local discomfort in the face and jaw, and none were classified as serious. In an intention-to-treat analysis of the main efficacy outcome, a statistically significant reduction of moderate and severe headache days in baseline versus month 2 was observed (16.3 ± 6.2 days baseline versus 7.6 ± 7.6 days month 2, p = 0.009). Eight out of 10 patients experienced an at least 50% reduction of moderate and severe headache days compared to baseline. Conclusion The result warrants randomised, placebo-controlled studies to establish both safety and efficacy of this potential novel treatment of chronic migraine.

Long-term spinal cord stimulation modifies canine intrinsic cardiac neuronal properties and ganglionic transmission during high-frequency repetitive activation.

Long-term spinal cord stimulation (SCS) applied to cranial thoracic SC segments exerts antiarrhythmic and cardioprotective actions in the canine heart in situ. We hypothesized that remodeling of intrinsic cardiac neuronal and synaptic properties occur in canines subjected to long-term SCS, specifically that synaptic efficacy may be preferentially facilitated at high presynaptic nerve stimulation frequencies. Animals subjected to continuous SCS for 5-8 weeks (long-term SCS: n = 17) or for 1 h (acute SCS: n = 4) were compared with corresponding control animals (long-term: n = 15, acute: n = 4). At termination, animals were anesthetized, the heart was excised and neurones from the right atrial ganglionated plexus were identified and studied in vitro using standard intracellular microelectrode technique. Main findings were as follows: (1) a significant reduction in whole cell membrane input resistance and acceleration of the course of AHP decay identified among phasic neurones from long-term SCS compared with controls, (2) significantly more robust synaptic transmission to rundown in long-term SCS during high-frequency (10-40 Hz) presynaptic nerve stimulation while recording from either phasic or accommodating postsynaptic neurones; this was associated with significantly greater posttrain excitatory postsynaptic potential (EPSP) numbers in long-term SCS than control, and (3) synaptic efficacy was significantly decreased by atropine in both groups. Such changes did not occur in acute SCS In conclusion, modification of intrinsic cardiac neuronal properties and facilitation of synaptic transmission at high stimulation frequency in long-term SCS could improve physiologically modulated vagal inputs to the heart.

Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons.

Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca(2+) markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca(2+) was fully inhibited by removal of extracellular Na(+), indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration-inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca(2+)/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca(2+) from intracellular Ca(2+) stores and calmodulin. The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

Calcium signals and FGF-2 induced neurite growth in cultured parasympathetic neurons: spatial localization and mechanisms of activation.

The growth of neuritic processes in developing neurons is tightly controlled by a wide set of extracellular cues that act by initiating downstream signaling cascades, where calcium signals play a major role. Here we analyze the calcium dependence of the neurite growth promoted by basic fibroblast growth factor (bFGF or FGF-2) in chick embryonic ciliary ganglion neurons, taking advantage of dissociated, organotypic, and compartmentalized cultures. We report that signals at both the growth cone and the soma are involved in the promotion of neurite growth by the factor. Blocking calcium influx through L- and N-type voltage-dependent calcium channels and transient receptor potential canonical (TRPC) channels reduces, while release from intracellular stores does not significantly affect, the growth of neuritic processes. Simultaneous recordings of calcium signals elicited by FGF-2 at the soma and at the growth cone show that the factor activates different patterns of responses in the two compartments: steady and sustained responses at the former, oscillations at the latter. At the soma, both voltage-dependent channel and TRPC blockers strongly affect steady-state levels. At the growth cone, the changes in the oscillatory pattern are more complex; therefore, we used a tool based on wavelet analysis to obtain a quantitative evaluation of the effects of the two classes of blockers. We report that the oscillatory behavior at the growth cone is dramatically affected by all the blockers, pointing to a role for calcium influx through the two classes of channels in the generation of signals at the leading edge of the elongating neurites.

Long-term estradiol-17ß administration changes the population of paracervical ganglion neurons supplying the ovary in adult gilts.

The aim of this study was to determine the influence of estradiol-17ß (E(2)) overdose on the number and distribution of ovarian parasympathetic neurons in the paracervical ganglion (PCG) in adult pigs. To identify the neurons innervating gonads on day 3 of the estrous cycle, the ovaries of both the control and experimental gilts were injected with retrograde neuronal tracer Fast Blue. From next day to the expected day 20 of the second studied cycle, experimental gilts were injected with E(2), while control gilts received oil. The PCG were then collected and processed for double-labeling immunofluorescence. Injections of E(2) increased the E(2) level in the peripheral blood approximately four- to fivefold and reduced the following in the PCG: the total number of Fast Blue-positive neurons; the number of perikarya in the lateral part of the PCG; the numbers of vesicular acetylcholine transporter (VAChT)(+)/somatostatin(+), VAChT(+)/vasoactive intestinal polypeptide (VIP)(+), VAChT(+)/neuronal isoform of nitric oxide synthase(+), VAChT(+)/VIP(-), VAChT(+)/dopamine ß-hydroxylase (DßH)(-), VAChT(-)/VIP(-), and VAChT(-)/DßH(-) perikarya; and the total number of perikarya expressing estrogen receptors (ERs) subtype α and/or ß. In summary, long-term E(2) treatment of adult gilts downregulates the population of both cholinergic and ERs expressing the PCG ovary-projecting neurons. Our results suggest that elevated E(2) levels occurring during pathological states may regulate gonadal function(s) by affecting ovary-supplying neurons.

Effect of onabotulinumtoxinA on intramural parasympathetic ganglia: an experimental study in the guinea pig bladder.

PURPOSE: We investigated whether onabotulinumtoxinA injected in the bladder would affect preganglionic parasympathetic nerve endings in intramural ganglia. MATERIALS AND METHODS: Guinea pig bladders were injected with 5 U of botulinum toxin. At 24 hours bladders were collected and processed for immunohistochemistry using tyrosine hydroxylase, and intact and cleaved SNAP-25. To identify the different populations of affected fibers coursing the ganglia we performed double immunoreactions for cleaved SNAP-25 and VAChT, TH or CGRP. RESULTS: VAChT immunoreactive fibers were identified in axons and varicosities of presynaptic to postganglionic parasympathetic neurons. Those fibers were also immunoreactive to SV2 and SNAP-25. The rare CGRP and TH immunoreactive fibers coursing in the ganglia did not express SV2 or SNAP-25. After onabotulinumtoxinA injection the cleaved form of SNAP-25 was abundantly expressed in parasympathetic fibers. CONCLUSIONS: Botulinum toxin injection in the bladder wall affects preganglionic parasympathetic nerve terminals. This could contribute to the strong effect of botulinum toxin on bladder smooth muscle activity.

Nitrergic nerves derived from the pterygopalatine ganglion innervate arteries irrigating the cerebrum but not the cerebellum and brain stem in monkeys.

The functional roles of the nitrergic nerves innervating the monkey cerebral artery were evaluated in a tension-response study examining isolated arteries in vitro and cerebral angiography in vivo. Nicotine produced relaxation of arteries by stimulation of nerve terminals innervating isolated monkey arteries irrigating the cerebrum, cerebellum and brain stem. Relaxation of arteries induced by nicotine was abolished by treatment with N(G)-nitro-L-arginine, a nitric oxide synthase inhibitor, and was restored by addition of L-arginine. Cerebral angiography showed that electrical stimulation of the unilateral greater petrosal nerve, which connects to the pterygopalatine ganglion via the parasympathetic ganglion synapse, produced vasodilatation of the anterior, middle and posterior cerebral arteries in the stimulated side. However, stimulation failed to produce vasodilatation of the superior and anterior-inferior cerebellar arteries and the basilar artery in anesthetized monkeys. Therefore, nitrergic nerves derived from the pterygopalatine ganglion appear to regulate cerebral vasomotor function. In contrast, circulation in the cerebellum and brain stem might be regulated by nitrergic nerves originating not from the pterygopalatine ganglion, but rather from an unknown ganglion (or ganglia).

Semaphorin 3A inhibits growth of adult sympathetic and parasympathetic neurones via distinct cyclic nucleotide signalling pathways.

BACKGROUND AND PURPOSE: Semaphorin 3A (Sema3A) is an important secreted repulsive guidance factor for many developing neurones. Sema3A continues to be expressed in adulthood, and expression of its receptor, neuropilin-1 (Nrp-1), can be altered by nerve injury. Autonomic neurones innervating the pelvic viscera are particularly susceptible to damage during pelvic surgical procedures, and failure to regenerate or aberrant growth of sympathetic and parasympathetic nerves lead to organ dysfunction. However, it is not known if adult pelvic neurones are potential targets for Sema3A. EXPERIMENTAL APPROACH: The effects of Sema3A and activation or inhibition of cyclic nucleotide signalling were assessed in adult rat pelvic ganglion neurones in culture using a growth cone collapse assay. KEY RESULTS: Sema3A caused growth cone collapse in both parasympathetic and sympathetic neurones expressing Nrp-1. However, the effect of Sema3A was mediated by distinct cyclic nucleotide signalling pathways in each neurone type. In parasympathetic neurones, cAMP and downstream activation of protein kinase A were required for growth cone collapse. In sympathetic neurones, cGMP was required for Sema3A-induced collapse; cAMP can also cause collapse but was not required. Sema3A-mediated, cGMP-dependent collapse in sympathetic neurones may require activation of cyclic nucleotide-gated ion channels (CNGCs). CONCLUSIONS AND IMPLICATIONS: We propose that Sema3A is an important guidance factor for adult pelvic autonomic neurones, and that manipulation of their distinct signalling mechanisms could potentially promote functional selective regeneration or attenuate aberrant growth. To our knowledge, this is also the first study to implicate CNGCs in regulating growth cone dynamics of adult neurones.

Benzalkonium chloride-treated anorectums mimicked endothelin-3-deficient aganglionic anorectums on manometry.

BACKGROUND/PURPOSE: The anorectal spasticity in Hirschsprung disease may be caused by the absence of enteric ganglia and/or the presence of hypertrophic nerves. Anorectal manometry of chemically denervated rectums was compared with that of congenital aganglionic rectums that also possessed hypertrophic nerves. METHODS: Aganglionic and ganglionic littermates were produced from breeding heterozygous lethal-spotted mice. Benzalkonium chloride was endorectally injected into ganglionic rectums to ablate the neural elements. Anorectal manometry was performed before the injection and on day 14 postinjection. The anorectal resting pressure was calculated based on the manometric tracing. Rectums were retrieved on day 14 for histologic evaluations. RESULTS: Benzalkonium chloride injection successfully ablated the rectal ganglia. Although ganglionic littermates exhibited regular slow waves on anorectal manometry, aganglionic lethal-spotted mice showed irregular waves. Similar to lethal spotted mice, benzalkonium chloride-treated mice exhibited significantly higher anorectal resting pressure than that of ganglionic mice. The slow waves were absent in benzalkonium chloride-treated mice. CONCLUSION: Benzalkonium chloride treatment produced aganglionic rectums that had higher resting pressure similar to the congenital aganglionic rectums. This suggests that hypertrophic nerves in congenital aganglionosis are not necessary to produce the anorectal spasticity.

Responses induced by acetylcholine and ATP in the rabbit petrosal ganglion.

Acetylcholine and ATP appear to mediate excitatory transmission between receptor (glomus) cells and the petrosal ganglion (PG) neuron terminals in the carotid body. In most species these putative transmitters are excitatory, while inhibitory effects had been reported in the rabbit. We studied the effects of the application of acetylcholine and ATP to the PG on the carotid nerve activity in vitro. Acetylcholine and ATP applied to the PG increased the carotid nerve activity in a dose-dependent manner. Acetylcholine-induced responses were mimicked by nicotine, antagonized by hexamethonium, and enhanced by atropine. Bethanechol had no effect on basal activity, but reduced acetylcholine-induced responses. Suramin antagonized ATP-induced responses, and AMP had little effect on the carotid nerve activity. Our results suggest that rabbit PG neurons projecting through the carotid nerve are endowed with nicotinic acetylcholine and purinergic P2 receptors that increase the carotid nerve activity, while simultaneous activation of muscarinic cholinergic receptors reduce the maximal response evoked by nicotinic cholinergic receptor activation.

Natural compounds endowed with cholinergic or anticholinergic activity. Enhancement of acetylcholine release by a quaternary derivative of L-hyoscyamine.

New compounds that target nicotinic receptors (nAChRs) have been sought to correct disorders affecting cholinergic transmission in central and peripheral synapses. A quaternary derivate of l-hyoscyamine, phenthonium (Phen), was shown by our group to enhance the spontaneous acetylcholine (ACh) release without altering the nerve-induced transmitter release at the neuromuscular junction. The effect was unrelated to membrane depolarization, and was not induced by an increase of calcium influx into the nerve terminal. Phen also presented a competitive antimuscarinic activity and blocked noncompetitively the neuromuscular transmission. In this work we re-examined the mechanisms underlying the facilitatory actions of Phen on [(3)H]-ACh release in isolated ganglia of the guinea pig ileal myenteric plexus. Exposure of the preparations to Phen (10-50 microM) increased the release of [(3)H]-ACh by 81 to 68% over the basal. The effect was not affected by the ganglionic nAChR antagonist hexamethonium (1 nM) at a concentration that inhibited the increase of [(3)H]-ACh release induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP, 30 microM). Association of Phen (10 microM) with DMPP potentiated the facilitatory effect of Phen. [(3)H]-ACh release was not altered by the muscarinic antagonists atropine (1 nM) or pirenzepine (1 microM). However, both antagonists inhibited the release of [(3)H]-ACh induced by either the muscarinic M1 agonist McN-343 (10 microM) or Phen (20 microM). The facilitatory effect of Phen was not altered by CdCl(2) (50 mM), but it was potentiated in the presence of tetraethylammonium (40 mM). The results indicate that the facilitatory action of Phen appears to be mediated by an increase of the inwardly rectifying potassium channels conductance probably related to the compound antimuscarinic activity.

Muscarinic acetylcholine receptor modulation of mu (mu) opioid receptors in adult rat sphenopalatine ganglion neurons.

The sphenopalatine ganglion (SPG) neurons represent the parasympathetic branch of the autonomic nervous system involved in controlling cerebral blood flow. In the present study, we examined the coupling mechanism between mu (mu) opioid receptors (MOR) and muscarinic acetylcholine receptors (mAChR) with Ca(2+) channels in acutely dissociated adult rat SPG neurons. Successful MOR activation was recorded in approximately 40-45% of SPG neurons employing the whole cell variant of the patch-clamp technique. In addition, immunofluorescence assays indicated that MOR are not expressed in all SPG neurons while M(2) mAChR staining was evident in all neurons. The concentration-response relationships generated with morphine and [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) showed IC(50) values of 15.2 and 56.1 nM and maximal Ca(2+) current inhibition of 26.0 and 38.7%, respectively. Activation of MOR or M(2) mAChR with morphine or oxotremorine-methiodide (Oxo-M), respectively, resulted in voltage-dependent inhibition of Ca(2+) currents via coupling with Galpha(i/o) protein subunits. The acute prolonged exposure (10 min) of neurons to morphine or Oxo-M led to the homologous desensitization of MOR and M(2) mAChR, respectively. The prolonged stimulation of M(2) mAChR with Oxo-M resulted in heterologous desensitization of morphine-mediated Ca(2+) current inhibition, and was sensitive to the M(2) mAChR blocker methoctramine. On the other hand, when the neurons were exposed to morphine or DAMGO for 10 min, heterologous desensitization of M(2) mAChR was not observed. These results suggest that in rat SPG neurons activation of M(2) mAChR likely modulates opioid transmission in the brain vasculature to adequately maintain cerebral blood flow.