Injection Of Carbachol Research Articles

Defective parasympathetic innervation is associated with airway branching abnormalities in experimental CDH.

Developmental mechanisms leading to lung hypoplasia in congenital diaphragmatic hernia (CDH) remain poorly defined. Pulmonary innervation is defective in the human disease and in the rodent models of CDH. We hypothesize that defective parasympathetic innervation may contribute to airway branching abnormalities and, therefore, lung hypoplasia, during lung development in CDH. The murine nitrofen model of CDH was utilized to study the effect of the cholinergic agonist carbachol on embryonic day 11.5 (E11.5) lung explant cultures. Airway branching and contractions were quantified. In a subset of experiments, verapamil was added to inhibit airway contractions. Sox9 immunostaining and 5-bromo-2-deoxyuridine incorporation were used to identify and quantify the number and proliferation of distal airway epithelial progenitor cells. Intra-amniotic injections were used to determine the in vivo effect of carbachol. Airway branching and airway contractions were significantly decreased in nitrofen-treated lungs compared with controls. Carbachol resulted in increased airway contractions and branching in nitrofen-treated lungs. Nitrofen-treated lungs exhibited an increased number of proliferating Sox9-positive distal epithelial progenitor cells, which were decreased and normalized by treatment with carbachol. Verapamil inhibited the carbachol-induced airway contractions in nitrofen-treated lungs but had no effect on the carbachol-induced increase in airway branching, suggesting a direct carbachol effect independent of airway contractions. In vivo treatment of nitrofen-treated embryos via amniotic injection of carbachol at E10.5 resulted in modest increases in lung size and branching at E17.5. These results suggest that defective parasympathetic innervation may contribute to airway branching abnormalities in CDH.

The interrelationship between cholinergic pathway in the magnocellular paraventricular nucleus and natriuresis.

The central nervous system is known to play important roles in the regulation of renal sodium excretion. The present study was designed to reveal the interrelationship between cholinergic pathway in the magnocellular paraventricular nucleus (PVN) and the natriuresis induced by brain cholinergic stimuli. The results indicated that urinary sodium excretion was significantly increased at 40 min after intracerebroventricular (ICV) injection of carbachol (CBC). Immunohistochemical studies showed that CBC increased choline acetyltransferase-immunoreactivity (ChAT-IR) in the magnocellular PVN and renal proximal convoluted tubule (PCT), respectively. After pretreatment with atropine, urinary sodium excretion was significantly reduced, and carbachol-increased ChAT-IR in the magnocellular PVN and PCT was also significantly decreased. These results suggested that brain cholinergic stimuli induced the natriuresis and increased the activity of cholinergic neurons in the magnocellular PVN and cholinergic system in the PCT. The blockade of muscarinic receptor completely abolished the natriuresis and partially inhibited carbachol-exerted stimulatory effects in the magnocellular PVN and PCT. To summarize, brain cholinergic pathway and peripheral cholinergic system in kidney were found to contribute to the natriuresis following brain cholinergic stimulation. Our findings revealed novel evidence that PVN was involved in the natriuresis via humoral mechanisms.

Critical role of cholinergic transmission from the laterodorsal tegmental nucleus to the ventral tegmental area in cocaine-induced place preference

Conditioned place preference (CPP) is widely used to investigate the rewarding properties of cocaine. Various brain regions and neurotransmitters are involved in developing cocaine CPP. However, the contribution of cholinergic transmission in the ventral tegmental area (VTA) to cocaine CPP remains largely unexplored. Here, we examined the role of cholinergic input arising from the laterodorsal tegmental nucleus (LDT) to the VTA in the acquisition and expression of cocaine CPP in rats. Intra-LDT injection of carbachol, which hyperpolarizes LDT neurons, and of NMDA and AMPA receptor antagonists before cocaine conditioning blocked and attenuated cocaine CPP, respectively, indicating the necessity of LDT activity for acquiring the CPP. Additionally, intra-VTA injection of scopolamine or mecamylamine before cocaine conditioning also attenuated cocaine CPP, demonstrating the contribution of cholinergic transmission via muscarinic and nicotinic acetylcholine receptors in CPP acquisition. Furthermore, intra-VTA injection of scopolamine or mecamylamine immediately before the test attenuated cocaine CPP, indicating that cholinergic signaling is also associated with the expression of CPP. These results suggest that cholinergic transmission from the LDT to the VTA is critically involved in both acquiring and retrieving cocaine-associated memories in cocaine CPP.

Carbachol excites sublaterodorsal nucleus neurons projecting to the spinal cord

Considerable electrophysiological and pharmacological evidence has long suggested an important role for acetylcholine in the regulation of rapid-eye-movement (REM) sleep. For example, injection of the cholinergic agonist carbachol into the dorsomedial pons produces an REM sleep-like state with muscle atonia and cortical activation, both of which are cardinal features of REM sleep. Located within this region of the pons is the sublaterodorsal nucleus (SLD), a structure thought to be both necessary and sufficient for generating REM sleep muscle atonia. Subsets of glutamatergic SLD neurons potently contribute to motor inhibition during REM sleep through descending projections to motor-related glycinergic/GABAergic neurons in the spinal cord and ventromedial medulla. Prior electrophysiological and pharmacological studies examining the effects of acetylcholine on SLD neurons have, however, produced conflicting results. In the present study, we sought to clarify how acetylcholine influences the activity of spinally projecting SLD (SLDsp) neurons. We used retrograde tracing in combination with patch-clamp recordings and recorded pre- and postsynaptic effects of carbachol on SLDsp neurons. Carbachol acted presynaptically by increasing the frequency of glutamatergic miniature excitatory postsynaptic currents. We also found that carbachol directly excited SLDsp neurons by activating an Na(+)-Ca(2+) exchanger. Both pre- and postsynaptic effects were mediated by co-activation of M1 and M3 muscarinic receptors. These observations suggest that acetylcholine produces synergistic, excitatory pre- and postsynaptic responses on SLDsp neurons that, in turn, probably serve to promote muscle atonia during REM sleep.

Separating analgesia from reward within the ventral tegmental area

Activation of the dopaminergic mesolimbic reward circuit that originates in the ventral tegmental area (VTA) is postulated to preferentially suppress emotional responses to noxious stimuli, and presumably contributes to the addictive liability of strong analgesics. VTA dopamine neurons are activated via cholinergic afferents and microinjection of carbachol (cholinergic agonist) into VTA is rewarding. Here, we evaluated regional differences within VTA in the capacity of carbachol to suppress rats’ affective response to pain (vocalization afterdischarges, VADs) and to support conditioned place preference (CPP) learning. As carbachol is a non-specific agonist, muscarinic and nicotinic receptor involvement was assessed by administering atropine (muscarinic antagonist) and mecamylamine (nicotinic antagonist) into VTA prior to carbachol treatment. Unilateral injections of carbachol (4μg) into anterior VTA (aVTA) and posterior VTA (pVTA) suppressed VADs and supported CPP; whereas, injections into midVTA failed to effect either VADs or CPP. These findings corroborate the hypothesis that the neural substrates underlying affective analgesia and reward overlap. However, the extent of the overlap was only partial. Whereas both nicotinic and muscarinic receptors contributed to carbachol-induced affective analgesia in aVTA, only muscarinic receptors mediated the analgesic action of carbachol in pVTA. The rewarding effects of carbachol are mediated by the activation of both nicotinic and muscarinic receptors in both aVTA and pVTA. The results indicate that analgesia and reward are mediated by separate cholinergic mechanisms within pVTA. Nicotinic receptor antagonism within pVTA failed to attenuate carbachol-induced analgesia, but prevented carbachol-induced reward. As addictive liability of analgesics stem from their rewarding properties, the present findings suggest that these processes can be neuropharmacologically separated within pVTA.

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

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

Dynamic Interaction of Spindles and Gamma Activity during Cortical Slow Oscillations and Its Modulation by Subcortical Afferents

Slow oscillations are a hallmark of slow wave sleep. They provide a temporal framework for a variety of phasic events to occur and interact during sleep, including the expression of high-frequency oscillations and the discharge of neurons across the entire brain. Evidence shows that the emergence of distinct high-frequency oscillations during slow oscillations facilitates the communication among brain regions whose activity was correlated during the preceding waking period. While the frequencies of oscillations involved in such interactions have been identified, their dynamics and the correlations between them require further investigation. Here we analyzed the structure and dynamics of these signals in anesthetized rats. We show that spindles and gamma oscillations coexist but have distinct temporal dynamics across the slow oscillation cycle. Furthermore, we observed that spindles and gamma are functionally coupled to the slow oscillations and between each other. Following the activation of ascending pathways from the brainstem by means of a carbachol injection in the pedunculopontine nucleus, we were able to modify the gain in the gamma oscillations that are independent of the spindles while the spindle amplitude was reduced. Furthermore, carbachol produced a decoupling of the gamma oscillations that are dependent on the spindles but with no effect on their amplitude. None of the changes in the high-frequency oscillations affected the onset or shape of the slow oscillations, suggesting that slow oscillations occur independently of the phasic events that coexist with them. Our results provide novel insights into the regulation, dynamics and homeostasis of cortical slow oscillations.

Effects of muscarinic agents on chick choroids in intact eyes and eyecups: evidence for a muscarinic mechanism in choroidal thinning

In chicks, ocular growth inhibition is associated with choroidal thickening and growth stimulation with choroidal thinning, suggesting a mechanistic link between the two responses. Because muscarinic antagonists inhibit the development of myopia in animal models by a non-accommodative mechanism, we tested the hypothesis that agonists would stimulate eye growth and thin the choroid. We also hypothesized that the effective growth-inhibiting antagonists would thicken the choroid. Chicks, age 12-16 days, were used. In vivo: Agonists: Single intravitreal injections (20 μL) of oxotremorine (oxo), pilocarpine (pilo), carbachol (carb), or arecaidine (arec) were given to otherwise untreated eyes. A-scan ultrasonography was done prior to injections, and at 3, 24, 48 and 72 h. Antagonists: -10D lenses were worn on one eye for 4 days. Atropine (atro), pirenzepine (pirz), oxyphenonium (oxy) or dicyclomine (dicy) were injected (20 μL) daily into lens-wearing eyes; saline injections were done as controls. Ultrasonography was done on d1 and on d4; on d4 measurements were done before and 3 h after injections. In vitro: Paired eyecups of retinal pigment epithelium (RPE), choroid and sclera were made from 1-week old chicks. All drugs except atropine were tested on one eyecup, its pair in plain medium. Choroidal thickness was measured at various times over 48 h. Agonists: In vivo, oxotremorine caused an increase in the rate of axial elongation (drug vs saline: 24-72 h: 338 μm vs 250 μm; p < 0.001). All except pilocarpine caused choroidal thinning by 24 h (oxo, carb and arec vs saline: -25, -35 and -46 μm vs 3 μm). In vitro, all agonists thinned choroids by 24 h (oxo: -6 vs 111 μm; pilo: 45 vs 212 μm; carb: -58 vs 65 μm; arec: 47 vs 139 μm; p < 0.05). Antagonists: Atropine, pirenzepine and oxyphenonium inhibited the development of myopia in negative lens-wearing eyes, and also caused choroidal thickening (drug vs saline: 42, 80, 88 vs 10 μm per 3 h). In vitro, pirenzepine thickened choroids by 3 h (77 vs 2 μm, p < 0.01). Muscarinic agonists caused choroidal thinning in intact eyes and eyecups, supporting a role for acetylcholine in the choroidal response to hyperopic defocus or form deprivation. Only oxotremorine stimulated eye growth, which is inconsistent with a muscarinic receptor mechanism for antagonist-induced eye growth inhibition. The dissociation between choroidal thinning and ocular growth stimulation for the other agonists in vivo suggest separate pathways for the two.

Evidence That Adrenergic Ventrolateral Medullary Cells Are Activated whereas Precerebellar Lateral Reticular Nucleus Neurons Are Suppressed during REM Sleep

Rapid eye movement sleep (REMS) is generated in the brainstem by a distributed network of neurochemically distinct neurons. In the pons, the main subtypes are cholinergic and glutamatergic REMS-on cells and aminergic REMS-off cells. Pontine REMS-on cells send axons to the ventrolateral medulla (VLM), but little is known about REMS-related activity of VLM cells. In urethane-anesthetized rats, dorsomedial pontine injections of carbachol trigger REMS-like episodes that include cortical and hippocampal activation and suppression of motoneuronal activity; the episodes last 4–8 min and can be elicited repeatedly. We used this model to determine whether VLM catecholaminergic cells are silenced during REMS, as is typical of most aminergic neurons studied to date, and to investigate other REMS-related cells in this region. In 18 anesthetized, paralyzed and artificially ventilated rats, we obtained extracellular recordings from VLM cells when REMS-like episodes were elicited by pontine carbachol injections (10 mM, 10 nl). One major group were the cells that were activated during the episodes (n = 10). Their baseline firing rate of 3.7±2.1 (SD) Hz increased to 9.7±2.1 Hz. Most were found in the adrenergic C1 region and at sites located less than 50 µm from dopamine β-hydroxylase-positive (DBH+) neurons. Another major group were the silenced or suppressed cells (n = 35). Most were localized in the lateral reticular nucleus (LRN) and distantly from any DBH+ cells. Their baseline firing rates were 6.8±4.4 Hz and 15.8±7.1 Hz, respectively, with the activity of the latter reduced to 7.4±3.8 Hz. We conclude that, in contrast to the pontine noradrenergic cells that are silenced during REMS, medullary adrenergic C1 neurons, many of which drive the sympathetic output, are activated. Our data also show that afferent input transmitted to the cerebellum through the LRN is attenuated during REMS. This may distort the spatial representation of body position during REMS.

Commissural NTS lesions enhance the pressor response to central cholinergic and adrenergic activation

Electrolytic lesions of the commissural nucleus of the solitary tract (commNTS) in rats enhance the pressor response to bilateral carotid occlusion or to intravenous infusion of hypertonic NaCl without changing baroreflex responses. In an opposite direction, commNTS lesions abolish the pressor responses to peripheral chemoreflex activation. These opposite effects of commNTS lesions apparently result from an impairment of sympathetic activation in one case and in a facilitation of vasopressin secretion in the others. In the present study, we investigated the effects of the electrolytic lesions of the commNTS in the pressor responses that depend on sympathetic activation and vasopressin secretion produced by central cholinergic or adrenergic activation with intracerebroventricular (i.c.v.) injections of carbachol or noradrenaline, respectively, in unanesthetized rats. Male Holtzman rats (280–320g, n=8–15/group) with acute (1 day) or chronic (21 days) sham or commNTS lesions (1mA×10s) and a stainless steel cannula implanted in the lateral ventricle were used. Acute commNTS lesions increased the pressor response to i.c.v. injection of carbachol (0.5nmol/1μ1) (52±2, vs. sham: 37±2mmHg) or noradrenaline (80nmol/1μl) (45±6, vs. sham: 30±3mmHg), whereas chronic commNTS lesions did not affect the pressor responses to the same treatments. Lesions of the commNTS impaired chemoreflex responses produced by intravenous KCN, without changing baroreflex responses. The results suggest that commNTS-dependent inhibitory signals are involved in the modulation of the pressor responses to central cholinergic and adrenergic activation, probably limiting vasopressin secretion.

Inhibition of A5 Neurons Facilitates the Occurrence of REM Sleep-Like Episodes in Urethane-Anesthetized Rats: A New Role for Noradrenergic A5 Neurons?

When rapid eye movement (REM) sleep occurs, noradrenergic cells become silent, with the abolition of activity in locus coeruleus (LC) neurons seen as a key event permissive for the occurrence of REM sleep. However, it is not known whether silencing of other than LC noradrenergic neurons contributes to the generation of REM sleep. In urethane-anesthetized rats, stereotyped REM sleep-like episodes can be repeatedly elicited by injections of the cholinergic agonist, carbachol, into a discrete region of the dorsomedial pons. We used this preparation to test whether inhibition of ventrolateral pontine noradrenergic A5 neurons only, or together with LC neurons, also can elicit REM sleep-like effects. To silence noradrenergic cells, we sequentially injected the α2-adrenergic agonist clonidine (20–40 nl, 0.75 mM) into both A5 regions and then the LC. In two rats, successful bilateral clonidine injections into the A5 region elicited the characteristic REM sleep-like episodes (hippocampal theta rhythm, suppression of hypoglossal nerve activity, reduced respiratory rate). In five rats, bilateral clonidine injections into the A5 region and then into one LC triggered REM sleep-like episodes, and in two rats injections into both A5 and then both LC were needed to elicit the effect. In contrast, in three rats, uni- or bilateral clonidine injections only into the LC had no effect, and clonidine injections placed in another six rats outside of the A5 and/or LC regions were without effect. The REM sleep-like episodes elicited by clonidine had similar magnitude of suppression of hypoglossal nerve activity (by 75%), similar pattern of hippocampal changes, and similar durations (2.5–5.3 min) to the episodes triggered in the same preparation by carbachol injections into the dorsomedial pontine reticular formation. Thus, silencing of A5 cells may importantly enable the occurrence of REM sleep-like episodes, at least under anesthesia. This is a new role for noradrenergic A5 neurons.

Activity of cholinergic neurons in the laterodorsal tegmental nucleus during emission of 22 kHz vocalization in rats

It has been postulated that the ascending cholinergic tegmental system is responsible for the initiation of the aversive emotional state with a concomitant alarm vocalization in the rat. It is assumed that the activity of cholinergic neurons of the laterodorsal tegmental nucleus (LDT) will cause release of acetylcholine in the target areas and will initiate the emission of 22 kHz vocalizations. The goal of the present study was to test the hypothesis that the cholinergic neurons of the LDT increase their activity during emission of 22 kHz alarm calls. Vocalizations were induced by an air puff or by intrahypothalamic-preoptic injection of carbachol. The activity of the LDT cholinergic neurons was studied by a double histochemical labelling for choline acetyltransferase, as a marker of cholinergic somata, and for c-Fos protein, as a marker of cells with heighten metabolic activity. Both air puff stimulation and intracerebral carbachol induced comparable 22 kHz alarm vocalizations. The activity of neurons in the LDT was significantly higher during prolonged emission of 22 kHz alarm calls induced by air puff or injection of carbachol than in the non-vocalizing or low-vocalizing controls. There were approximately two times more of all c-Fos-labelled cells in the LDT of vocalizing animals and 2.5 times more active cholinergic neurons during prolonged 22 kHz vocalization than in the control conditions without vocalization. However, the active cholinergic neurons constituted only a small proportion of all active LDT cells (2.3%). At the same time, there were no significant increases in the number of c-Fos-labelled cells in the neighbouring pedunculopontine nucleus (PPT). These findings lead to the conclusion that the neurons of the LDT, including cholinergic neurons, but not those of the PPT, significantly increased their activity during prolonged emission of alarm vocalizations, as evidenced by the c-Fos immunoreactivity.

Cardiovascular responses in rats with chronic renal failure

Cardiovascular responses were determined in rats with chronic renal failure (CRF) produced by five sixths nephrectomy and in sham-operated rats. The conscious systolic blood pressure of rats with CRF was significantly higher than the pressure in controls although, after anaesthesia, there were no significant differences in the mean arterial pressure between the two groups of rats. The pressor responses to noradrenaline in rats with CRF were not significantly different from those recorded in sham-operated controls. The bradycardia elicited by electrical stimulation of the vagus nerve was significantly diminished in rats with CRF. However, indomethacin treatment (1 mg kg-1 s.c. twice daily for 2 days) abolished the differences in response to vagal stimulation. Changes in heart rate in response to electrical stimulation of the cervical sympathetic nerve and to bolus i.v. injections of isoprenaline and carbachol were similar in rats with CRF and controls. The most notable disturbance of cardiovascular function in rats with CRF is the diminished cardiac chronotropic response to vagal stimulation which appears to be mediated by a presynaptic action of prostaglandins.

Functional interaction between medial thalamus and rostral anterior cingulate cortex in the suppression of pain affect

The medial thalamic parafascicular nucleus (PF) and the rostral anterior cingulate cortex (rACC) are implicated in the processing and suppression of the affective dimension of pain. The present study evaluated the functional interaction between PF and rACC in mediating the suppression of pain affect in rats following administration of morphine or carbachol (acetylcholine agonist) into PF. Vocalizations that occur following a brief noxious tailshock (vocalization afterdischarges) are a validated rodent model of pain affect, and were preferentially suppressed by injection of morphine or carbachol into PF. Vocalizations that occur during tailshock were suppressed to a lesser degree, whereas, spinal motor reflexes (tail flick and hindlimb movements) were only slightly suppressed by injection of carbachol into PF and unaffected by injection of morphine into PF. Blocking glutamate receptors in rACC (NMDA and non-NMDA) by injecting d-2-amino-5-phosphonovalerate (AP-5) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) produced dose-dependent antagonism of morphine-induced increases in vocalization thresholds. Carbachol-induced increases in vocalization thresholds were not affected by injection of either glutamate receptor antagonist into rACC. The results demonstrate that glutamate receptors in the rACC contribute to the suppression of pain affect produced by injection of morphine into PF, but not to the suppression of pain affect generated by intra-PF injection of carbachol.

Role of PPARγ/Nitric Oxide Synthase Signaling in the Cyclosporine-induced Attenuation of Endothelium-dependent Renovascular Vasodilation

Evidence from our laboratory and others suggests a negative effect for cyclosporine A (CSA) on renovascular reactivity. This study investigated the role of peroxisome proliferator-activated receptor gamma (PPAR gamma)/nitric oxide synthase (NOS) signaling in the CSA-induced attenuation of endothelium-dependent vasodilations in phenylephrine-preconstricted perfused kidneys of rats. Bolus injection of carbachol (4 micromoL) reduced the renal perfusion pressure with a peak depressor effect observed at 2 minutes. CSA (5 microM) infusion significantly attenuated the vasodilatory action of carbachol. The specificity of this interaction was verified by the lack of effect of CSA on renal vasodilation caused by papaverine (50 nmol). The carbachol-induced renal vasodilations were also reduced after infusion of N-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor, 100 microM) or 2-chloro-5-nitro-N-phenylbenzamide (GW9662, PPAR gamma antagonist, 1 microM). The attenuation of carbachol vasodilation by CSA was abolished in presence of L-arginine or L-NAME in contrast to no effect for GW9662. Pioglitazone (PPAR gamma agonist, 10 microM) abolished the CSA-induced attenuation of carbachol responses, an effect that was not manifest in presence of GW9662 or l-NAME. These findings suggest that PPAR gamma act tonically to facilitate renovascular dilatory response to endothelial muscarinic receptor activation. More importantly, NOS signaling downstream of PPAR gamma mediates, at least partly, the inhibitory effect of CSA on carbachol vasodilations.

Pioglitazone Ameliorates Cyclosporine‐Induced Attenuation Of Carbachol Renovascular Vasodilations: Role Of PPARγ/Nitric Oxide Synthase Signaling

We previously reported that cyclosporine A (CSA) impairs reno‐vascular reactivity. This study investigated the role of peroxisome proliferator‐activated receptor‐gamma (PPARγ)/NOS signaling in the CSA‐induced attenuation of endothelium‐dependent vasodilations in phenylephrine‐preconstricted perfused kidneys of rats. Bolus injection of carbachol (4 μmol) reduced the renal perfusion pressure (RPP) with a peak depressor effect observed at 2 min. CSA (5 μM) infusion attenuated carbachol vasodilations. Similar inhibitory effects on carbachol vasodilations were evident after infusion of NG‐nitro‐L‐arginine methyl ester (L‐NAME, NOS inhibitor) or GW9662 (PPARγ antagonist). The CSA‐evoked attenuation of carbachol vasodilation was abolished in presence of L‐NAME in contrast to no effect for GW9662. Pioglitazone, PPARγ agonist, abolished the attenuation of these responses elicited by CSA; an effect that was not manifest in presence of GW9662. The specificity of the interaction with carbachol was verified by the lack of effect of CSA on vasodilations caused by papaverine. It is concluded that the interruption of PPARγ/NOS signaling mediates, at least partly, the inhibitory effect of CSA on renovascular responses to carbachol. Further, functional NOS is necessary for the amelioration by pioglitazone of the CSA‐carbachol interaction.

Carbachol injection into the pontine reticular formation depresses laryngeal muscle activities and airway reflexes in decerebrate cats

To understand the role of cholinoceptive, medial pontine reticular formation (mPRF) neurons in the control of upper airway, pharyngolaryngeal reflexes, we measured activities of intrinsic laryngeal muscles (posterior cricoarytenoid, PCA; thyroarytenoid, TA), diaphragm (DIA), genioglossus (GG) and a neck muscle (trapezius) in unanesthetized, decerebrated, spontaneously breathing cats with and without mPRF carbachol injections. The ethimoidal nerve was electrically stimulated to evoke sneezing, and the superior laryngeal nerve to evoke the laryngeal reflex, swallowing, and coughing. Carbachol reduced the amplitudes of the spontaneous electromyographic activities in the neck, TA, PCA, GG, and DIA to 7%, 30%, 54%, 45% and 71% of control, respectively, reduced the respiratory rate to 53% without changes in expiratory CO 2 concentration; the magnitude of the laryngeal reflex in the TA muscle to 56%; increased its latency by 13%; and reduced the probability of stimulus-induced sneezing, swallowing, and coughing to less than 40%. These changes lasted more than 1 h. These data demonstrate that important upper airway reflexes are suppressed by increasing cholinergic neurotransmission in the mPRF. Because acetylcholine release in the mPRF changes in accordance with sleep–wake cycles, the present findings are relevant to the control of upper airway reflexes during various vigilance states.

Cholinergic stimulation of the nucleus basalis of Meynert and reticular thalamic nucleus affects spike-and-wave discharges in WAG/Rij rats

The role of the cholinergic innervated nucleus basalis of Meynert (NB) and reticular thalamic nucleus (RT) in the generation or modulation of spontaneously occurring spike-and-wave discharges (SWDs) was investigated in the WAG/Rij rat model of absence epilepsy. The cholinergic agonist carbachol and the muscarinic antagonist scopolamine were injected in the NB and RT in the doses of 0.55 and 5.5 nmol while the EEG was recorded. Carbachol injected in the NB decreased the number and the mean duration of SWDs. Scopolamine alone had no influence on SWDs, but could antagonize the effects of carbachol if administered simultaneously in NB. Injections of carbachol in the RT inhibited the occurrence of SWDs, but did not affect the mean duration. Scopolamine administered in the RT had no influence on seizure activity. It is concluded that cholinergic stimulation of the NB or the RT inhibits the cortical synchronous activity characterizing SWDs.

Cholinergic Pairing with Visual Activation Results in Long-Term Enhancement of Visual Evoked Potentials

Acetylcholine (ACh) contributes to learning processes by modulating cortical plasticity in terms of intensity of neuronal activity and selectivity properties of cortical neurons. However, it is not known if ACh induces long term effects within the primary visual cortex (V1) that could sustain visual learning mechanisms. In the present study we analyzed visual evoked potentials (VEPs) in V1 of rats during a 4–8 h period after coupling visual stimulation to an intracortical injection of ACh analog carbachol or stimulation of basal forebrain. To clarify the action of ACh on VEP activity in V1, we individually pre-injected muscarinic (scopolamine), nicotinic (mecamylamine), α7 (methyllycaconitine), and NMDA (CPP) receptor antagonists before carbachol infusion. Stimulation of the cholinergic system paired with visual stimulation significantly increased VEP amplitude (56%) during a 6 h period. Pre-treatment with scopolamine, mecamylamine and CPP completely abolished this long-term enhancement, while α7 inhibition induced an instant increase of VEP amplitude. This suggests a role of ACh in facilitating visual stimuli responsiveness through mechanisms comparable to LTP which involve nicotinic and muscarinic receptors with an interaction of NMDA transmission in the visual cortex.

Carbachol injections in the nucleus pontis oralis elicit REM sleep‐like depression of XII nerve activity in the rat in situ perfused working heart‐brainstem preparation (WHBP)

Patients with obstructive sleep apnea suffer from nocturnal episodes of upper airway collapse and hypoxia that is most severe during rapid‐eye‐movement sleep (REMS). The XII activity is diminished and the upper airway muscle tone is reduced. To study the central neural mechanisms of REMS, in vivo pharmacological models have been used (Kubin, 2001). To test whether upper airway motor tone depression can be also elicited in the in situ rat WHBP, carbachol (25‐50 nl, 10mM) was injected into the pons in n=33 WHBPs while recording XII and phrenic nerve activities (PNA). In 23 preparations, depression of XII nerve activity lasting 16‐24 min occurred within 142 s ± 26. The depressant episodes could be elicited repeatedly by injections at the same site at &gt;30 min intervals. Histological analysis revealed effective injection sites in the nucleus pontis oralis, Bregma ‐8.16 to ‐8.76, (Paxinos and Watson, 2005). Ineffective injection sites were either located in the nucleus pontis caudalis, were too ventral or too lateral. Only carbachol injections into the nucleus pontis oralis trigger REMS like alterations of the breathing pattern in the WHBP.Funded by BCCN Göttingen and HL‐47600.