Postsynaptic muscarinic M1 receptors activate prefrontal cortical EEG of C57BL/6J mouse

Recent pharmacological studies exploring the functional roles of muscarinic cholinergic receptor (mAChR) subtypes in prefrontal cortex of C57BL/6J (B6) mouse have provided evidence for a presynaptic M2 autoreceptor. The B6 mouse was chosen for these studies because it is a genetically well-characterized model that also provides the genomic background for many genetically modified mice. In addition to increasing ACh release, one functional consequence of pharmacologically blocking the cortical M2 autoreceptor is activation of the contralateral prefrontal cortical EEG. To date, the mechanisms through which M2 autoreceptor antagonism causes cortical EEG activation have not been investigated. The present study tested the hypothesis that, in the B6 mouse, prefrontal cortical ACh activates the contralateral prefrontal EEG via postsynaptic M1 receptors. This hypothesis was tested in 15 mice using in vivo microdialysis delivery of muscarinic antagonists with simultaneous quantification of ACh release, number of 7- to 14-Hz EEG spindles, and fast Fourier transformation analysis of prefrontal EEG. Dialysis delivery of the nonsubtype selective muscarinic antagonist scopolamine (10 nM) significantly (P = 0.01) increased ACh release. Quantitative EEG analysis showed that scopolamine did not alter contralateral prefrontal cortical EEG. To differentiate mAChR subtypes mediating pre- versus postsynaptic responses, additional experiments used muscarinic antagonists with different affinities for the five mAChR subtypes. Microdialysis delivery of 3 nM AF-DX 116, a muscarinic antagonist with relatively high affinity for the M2 and M4 subtypes, significantly (P < 0.01) increased prefrontal cortical ACh release and activated EEG in the contralateral prefrontal cortex. EEG activation was characterized by a significant decrease in number of 7- to 14-Hz EEG spindles (P < 0.0001) and power (Vrms) of EEG slow waves (P < 0.05). Microdialysis delivery of 3 nM AF-DX 116 plus 3 nM pirenzepine, a relatively selective M1 and M4 muscarinic antagonist, also significantly (P < 0.01) increased ACh release but did not decrease the number of EEG spindles and did not change EEG slow waves. The differential EEG and ACh responses to dialysis delivery of the muscarinic antagonists support the conclusion that, in B6 mouse, postsynaptic muscarinic receptors of the M1 subtype are a primary site by which ACh activates the EEG.     

Prefrontal cortex acetylcholine release,EEG slow waves,and spindles are modulated by M2 autoreceptors in C57BL/6J mouse

Recent evidence suggests that muscarinic cholinergic receptors of the M2 subtype serve as autoreceptors modulating acetylcholine (ACh) release in prefrontal cortex. The potential contribution of M2 autoreceptors to excitability control of prefrontal cortex has not been investigated. The present study tested the hypothesis that M2 autoreceptors contribute to activation of the cortical electroencephalogram (EEG) in C57BL/6J (B6) mouse. This hypothesis was evaluated using microdialysis delivery of the muscarinic antagonist AF-DX116 (3 nM) while simultaneously quantifying ACh release in prefrontal cortex, number of 7- to 14-Hz EEG spindles, and EEG power spectral density. Mean ACh release in prefrontal cortex was significantly increased (P < 0.0002) by AF-DX116. The number of 7- to 14-Hz EEG spindles caused by halothane anesthesia was significantly decreased (P < 0.0001) by dialysis delivery of AF-DX116 to prefrontal cortex. The cholinergically induced cortical activation was characterized by a significant (P < 0.05) decrease in slow-wave EEG power. Together, these neurochemical and EEG data support the conclusion that M2 autoreceptor enhancement of ACh release in prefrontal cortex activates EEG in contralateral prefrontal cortex of B6 mouse. EEG slow-wave activity varies across mouse strains, and the results encourage comparative phenotyping of cortical ACh release and EEG in additional mouse models.     

Carbachol in the pontine reticular formation of C57BL/6J mouse decreases acetylcholine release in prefrontal cortex

The prefrontal cortex and brainstem modulate autonomic and arousal state control but the neurotransmitter mechanisms underlying communication between prefrontal cortex and brainstem remain poorly understood. This study examined the hypothesis that microdialysis delivery of carbachol to the pontine reticular formation (PRF) of anesthetized C57BL/6J (B6) mouse modulates acetylcholine (ACh) release in the frontal association cortex. Microdialysis delivery of carbachol (8.8 mM) to the PRF caused a significant (P<0.01) decrease (-28%) in ACh release in the frontal association cortex, a significant (P<0.01) decrease (-23%) in respiratory rate, and a significant (P<0.01) increase (223%) in time to righting after anesthesia. Additional in vitro studies used the [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) assay to test the hypothesis that muscarinic cholinergic receptors activate guanine nucleotide binding proteins (G proteins) in the frontal association cortex and basal forebrain. In vitro treatment with carbachol (1 mM) caused a significant (P<0.01) increase in [(35)S]GTPgammaS binding in the frontal association cortex (62%) and basal forebrain nuclei including medial septum (227%), vertical (210%) and horizontal (165%) limbs of the diagonal band of Broca, and substantia innominata (127%). G protein activation by carbachol was concentration-dependent and blocked by atropine, indicating that the carbachol-stimulated [(35)S]GTPgammaS binding was mediated by muscarinic cholinergic receptors. Together, the in vitro and in vivo data show for the first time in B6 mouse that cholinergic neurotransmission in the PRF can significantly alter ACh release in frontal association cortex, arousal from anesthesia, and respiratory rate.     

Ketamine and MK-801 decrease acetylcholine release in the pontine reticular formation,slow breathing,and disrupt sleep

STUDY OBJECTIVES: Ketamine induces a dissociated state of consciousness by binding to the phencyclidine binding site within the ion channel gated by the N-methyl-D-aspartate (NMDA) receptor. The brain regions and neurotransmitter systems mediating ketamine-induced alterations in arousal remain incompletely understood. This study used in vivo microdialysis to test the hypothesis that ketamine alters acetylcholine (ACh) release in the medial pontine reticular formation (mPRF). DESIGN: Acetylcholine (ACh) release, sleep, and breathing were quantified following systemic ketamine administration. Microdialysis was used to deliver the NMDA-channel blocker dizocilpine maleate (MK-801) and the R(-)-isomer of ketamine into the mPRF while measuring ACh release. SETTING: N/A PARTICIPANTS: N/A INTERVENTIONS: N/A MEASUREMENTS AND RESULTS: Systemically administered ketamine disrupted normal sleep-cycle organization, reduced mPRF ACh release, and significantly slowed rate of breathing. Dialysis delivery of MK-801 to the mPRF significantly decreased respiratory rate and mPRF ACh release. Dialysis delivery to the mPRF of the R(-)-ketamine isomer significantly decreased mPRF ACh release. CONCLUSIONS: Decreased mPRF ACh release caused by systemically administered ketamine was mimicked by mPRF dialysis delivery of MK-801 and the R(-)-ketamine isomer. These data are consistent with the conclusion that systemically administered ketamine may alter arousal and breathing, in part, by altering cholinergic neurotransmission in the mPRF.     

M2 muscarinic receptors in pontine reticular formation of C57BL/6J mouse contribute to rapid eye movement sleep generation

Microinjecting the acetylcholinesterase inhibitor neostigmine into the pontine reticular formation of C57BL/6J (B6) mouse causes a rapid eye movement (REM) sleep-like state. This finding is consistent with similar studies in cat and both sets of data indicate that the REM sleep-like state is caused by increasing levels of endogenous acetylcholine (ACh). Muscarinic cholinergic receptors have been localized to the pontine reticular formation of B6 mouse but no previous studies have examined which of the five muscarinic receptor subtypes participate in cholinergic REM sleep enhancement. This study examined the hypothesis that M2 receptors in pontine reticular formation of B6 mouse contribute to the REM sleep-like state caused by pontine reticular formation administration of neostigmine. B6 mice (n=13) were implanted with electrodes for recording states of sleep and wakefulness and with microinjection cannulae aimed for the pontine reticular formation. States of sleep and wakefulness were recorded for 4 h following pontine reticular formation injection of saline (control) or neostigmine. Experiments designed to gain insight into the muscarinic receptor subtypes mediating REM sleep enhancement involved pontine reticular formation administration of neostigmine after pertussis toxin, neostigmine after methoctramine, and neostigmine after pirenzepine. Pertussis toxin was used to block effects mediated by M2 and M4 receptors. Methoctramine was used to block M2 and M4 receptors, and pirenzepine was used to block M1 and M4 receptors. Pertussis toxin and methoctramine significantly decreased the neostigmine-induced REM sleep-like state. In contrast, pretreatment with pirenzepine did not significantly decrease the REM sleep-like state caused by neostigmine. These results support the interpretation that M2 receptors in the pontine reticular formation of B6 mouse contribute to the generation of REM sleep.     

Pontine and basal forebrain cholinergic interaction: implications for sleep and breathing

Pontine and forebrain cholinergic nuclei contribute to the regulation of breathing and arousal. This report summarizes experiments in rat (n = 20) concerning the cholinergic interaction between pons and basal forebrain. In vitro [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) autoradiography quantified carbachol-stimulated guanine nucleotide binding (G) protein activation in seven basal forebrain nuclei. Carbachol significantly increased [(35)S]GTPgammaS binding in the vertical and horizontal limbs of the diagonal band of Broca, medial and lateral septum, and nucleus basalis (B)/substantia innominata (SI). In vitro receptor autoradiography demonstrated muscarinic receptors in the same nuclei where carbachol caused G protein activation. In vivo experiments showed that carbachol administered to the pontine reticular formation (PnO) significantly decreased the number of 7-14Hz spindles in the electroencephalogram (EEG), decreased acetylcholine release in SI, and decreased respiratory rate. Carbachol microinjection into SI did not alter the number of EEG spindles or respiratory rate. The results help clarify that EEG and rate of breathing are more effectively modulated by cholinergic neurotransmission in PnO than in SI.     

Pontine reticular formation (PnO) administration of hypocretin-1 increases PnO GABA levels and wakefulness

STUDY OBJECTIVES: GABAergic transmission in the oral part of the pontine reticular formation (PnO) increases wakefulness. The hypothalamic peptide hypocretin-1 (orexin A) promotes wakefulness, and the PnO receives hypocretinergic input. The present study tested the hypothesis that PnO administration of hypocretin-1 increases PnO GABA levels and increases wakefulness. This study also tested the hypothesis that wakefulness is either increased or decreased, respectively, by PnO administration of drugs known to selectively increase or decrease GABA levels. DESIGN: Awithin-subjects design was used for microdialysis and microinjection experiments. SETTING: University of Michigan. PATIENTS OR PARTICIPANTS: Experiments were performed using adult male Crl:CD (SD)IGS BR (Sprague-Dawley) rats (n=46). INTERVENTIONS: PnO administration of hypocretin-1, nipecotic acid (a GABA uptake inhibitor that increases extracellular GABA levels), 3-mercaptopropionic acid (a GABA synthesis inhibitor that decreases extracellular GABA levels; 3-MPA), and Ringer solution (vehicle control). MEASUREMENTS AND RESULTS: Dialysis administration of hypocretin-1 to the PnO caused a statistically significant, concentration-dependent increase in PnO GABA levels. PnO microinjection of hypocretin-1 or nipecotic acid caused a significant increase in wakefulness and a significant decrease in non-rapid eye movement (NREM) sleep and REM sleep. Microinjecting 3-MPA into the PnO caused a significant increase in NREM sleep and REM sleep and a significant decrease in wakefulness. CONCLUSIONS: An increase or a decrease in PnO GABA levels causes an increase or decrease, respectively, in wakefulness. Hypocretin-1 may promote wakefulness, at least in part, by increasing GABAergic transmission in the PnO.     

Sleep and GABA levels in the oral part of rat pontine reticular formation are decreased by local and systemic administration of morphine

Morphine, a mu-opioid receptor agonist, is a commonly prescribed treatment for pain. Although highly efficacious, morphine has many unwanted side effects including disruption of sleep and obtundation of wakefulness. One mechanism by which morphine alters sleep and wakefulness may be by modulating GABAergic signaling in brain regions regulating arousal, including the pontine reticular nucleus, oral part (PnO). This study used in vivo microdialysis in unanesthetized Sprague-Dawley rat to test the hypothesis that mu-opioid receptors modulate PnO GABA levels. Validation of the high performance liquid chromatographic technique used to quantify GABA was obtained by dialyzing the PnO (n=4 rats) with the GABA reuptake inhibitor nipecotic acid (500 microM). Nipecotic acid caused a 185+/-20% increase in PnO GABA levels, confirming chromatographic detection of GABA and demonstrating the existence of functional GABA transporters in rat PnO. Morphine caused a concentration-dependent decrease in PnO GABA levels (n=25 rats). Coadministration of morphine (100 microM) with naloxone (1 microM), a mu-opioid receptor antagonist, blocked the morphine-induced decrease in PnO GABA levels (n=5 rats). These results show for the first time that mu-opioid receptors in rat PnO modulate GABA levels. A second group of rats (n=6) was used to test the hypothesis that systemically administered morphine also decreases PnO GABA levels. I.v. morphine caused a significant (P<0.05) decrease (19%) in PnO GABA levels relative to control i.v. infusions of saline. Finally, microinjections followed by 2 h recordings of electroencephalogram and electromyogram tested the hypothesis that PnO morphine administration disrupts sleep (n=8 rats). Morphine significantly (P<0.05) increased the percent of time spent in wakefulness (65%) and significantly (P<0.05) decreased the percent of rapid eye movement (REM) sleep (-53%) and non-REM sleep (-69%). The neurochemical and behavioral data suggest that morphine may disrupt sleep, at least in part, by decreasing GABAergic transmission in the PnO.     

Contraction and [3H]QNB binding in collagenase isolated fundic smooth muscle cells

Smooth muscle cells from the guinea pig gastric fundus were isolated by successive collagenase digestions. Tritiated quinuclidinyl benzilate [( 3H]QNB) was used to study the binding characteristics of the muscarinic cholinergic receptors on these cells. Each cell bound 8.3 X 10(-19) mol of QNB, and a concentration of QNB of 0.19 nM was required to label one-half of the binding sites. This suggests a concentration of about 500,000 muscarinic cholinergic receptors per smooth muscle cell. The muscarinic cholinergic receptor antagonists atropine and scopolamine inhibited QNB binding with a 50% inhibiting concentration (IC50) in the nanomolar range, whereas the agonists acetylcholine (ACh), oxotremorine, and carbamylcholine had IC50S in the micromolar range. Hill coefficients (nH) for antagonists approached unity, but agonists displayed fractional nH. Exposure of cells to cholinergic muscarinic agonists resulted in dose-dependent decreases in cell length. The concentration of agonist required to induce half-maximal contractions (ED50) was 8.3 X 10(-12) M for ACh and 6.3 X 10(-13) M for oxotremorine. Atropine (10(-9) M) decreased the sensitivity to ACh, increasing the ED50 for ACh-induced contractions to 1.2 X 10(-10) M. These results suggest the existence of muscarinic receptor heterogeneity for cholinergic agonists but not for antagonists.     

GABAA receptors inhibit acetylcholine release in cat pontine reticular formation: implications for REM sleep regulation

This study used in vivo microdialysis in cat (n=12) to test the hypothesis that gamma aminobutyric acid A (GABAA) receptors in the pontine reticular formation (PRF) inhibit acetylcholine (ACh) release. Animals were anesthetized with halothane to hold arousal state constant. Six concentrations of the GABAA receptor antagonist bicuculline (0.03, 0.1, 0.3, 1, 3, and 10 mM) were delivered to a dialysis probe in the PRF, and endogenously released ACh was collected simultaneously. Bicuculline caused a concentration dependent increase in ACh release (maximal increase=345%; EC50=1.3 mM; r2=0.997). Co-administration of the GABAA receptor agonist muscimol prevented the bicuculline-induced increase in ACh release. In a second series of experiments, the effects of bicuculline (0.1, 0.3, 1, and 3 mM) on ACh release were examined without the use of general anesthesia. States of wakefulness, rapid-eye-movement (REM) sleep, and non-REM sleep were identified polygraphically before and during dialysis delivery of bicuculline. Higher concentrations of bicuculline (1 and 3 mM) significantly increased ACh release during wakefulness (36%), completely suppressed non-REM sleep, and increased ACh release during REM sleep (143%). The finding that ACh release in the PRF is modulated by GABAA receptors is consistent with the interpretation that inhibition of GABAergic transmission in the PRF contributes to the generation of REM sleep, in part, by increasing pontine ACh release.     

Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition

The cholinergic neurons in the septohippocampal projection are implicated in hippocampal functions such as spatial learning and memory. The aim of this study was to examine how septohippocampal cholinergic transmission is modulated by muscarinic inputs and by the neuropeptide galanin, co-localized with acetylcholine (ACh) in septohippocampal cholinergic neurons, and how spatial learning assessed by the Morris water maze test is affected. Muscarinic inputs to the septal area are assumed to be excitatory, whereas galanin is hypothesized to inhibit septohippocampal cholinergic function. To test these hypotheses, compounds were microinjected into the medial septum and hippocampal ACh release was assessed by microdialysis probes in the ventral hippocampus of the rat. Blockade of septal muscarinic transmission by intraseptal scopolamine increased hippocampal ACh release suggesting that septal cholinergic neurons are under tonic inhibition. Stimulation of septal muscarinic receptors by carbachol also increased hippocampal ACh release. Despite this increase, both scopolamine and carbachol tended to impair hippocampus-dependent spatial learning. This finding also suggests a revision of the simplistic notion that an increase in hippocampal ACh may be facilitatory for learning and memory. Galanin infused into the medial septum enhanced hippocampal ACh release and facilitated spatial learning, suggesting that septal galanin, contrary to earlier claims, does not inhibit but excites septohippocampal cholinergic neurons. Galanin receptor stimulation combined with muscarinic blockade in the septal area resulted in an excessive increase of hippocampal ACh release combined with an impairment of spatial learning. This finding suggests that the level of muscarinic activity within the septal area may determine the effects of galanin on hippocampal cognitive functions. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.     

Presynaptic inhibition of acetylcholine release

High potassium (51 mM) has been shown to evoke release of acetylcholine ([3H]ACh and endogenous ACh) from cholinergic nerves in rat bronchial smooth muscle. The release of [3H]ACh was reduced by 85% when the Ca2+ concentration was changed from 2 to 0.1 mM. The veratridine-induced release was completely inhibited by tetrodotoxin, but tetrodotoxin did not reduce the potassium-evoked release. The muscarinic agonist, oxotremorine, reduced the potassium stimulated release of [3H]ACh, without affecting the basal release. In contrast, scopolamine substantially potentiated the potassium-evoked release. Adenosine had a dual effect in the rat bronchi. Adenosine inhibited the potassium-evoked release of [3H]ACh and this presynaptic effect of adenosine was antagonized by 8-phenyltheophylline. Adenosine also induced contraction of the bronchial smooth muscle and there was potentiation by adenosine of the ACh-induced contraction. The results indicate that cholinergic nerve terminals in the rat bronchi possess muscarinic receptors which inhibit the release of ACh. Adenosine may have analogous effects, e.g. presynaptic inhibition of transmitter release in addition to postsynaptic enhancement of bronchial smooth muscle contraction.     

Interaction of noradrenergic and cholinergic systems in regulation of ocular dominance plasticity

We studied interactions among the noradrenergic (NA) and the muscarinic cholinergic (ACh) systems in the regulation of ocular dominance plasticity in kitten visual cortex. The cortex was bilaterally infused with 6-hydroxydopamine (6-OHDA) for a week. Upon termination of the 6-OHDA infusion, one hemisphere was infused with a muscarinic ACh agonist, bethanechol, through the same, chronically implanted cannula for the second week together with monocular lid suture. The other hemisphere received an infusion of the vehicle solution alone. (1) Only in the hemisphere infused with bethanechol at relatively high concentrations did we obtain a clear shift in ocular dominance. We also found that the effect of bethanechol was concentration-dependent. (2) By comparing necessary concentrations of bethanechol and NA for the respective maximal effects, we noted that the former was at least 100-fold less effective than the latter in restoring the plasticity. (3) The cortical infusion of bethanechol did not restore the plasticity to the propranolol-pretreated cortex; the ocular dominance distribution remained virtually unchanged. This result was interpreted as suggesting that functioning beta-adrenoreceptors are needed for the cortical effect of activating the muscarinic ACh receptors to become detectable. (4) The expected shift in ocular dominance following monocular deprivation was partially suppressed, when highly concentrated scopolamine, a muscarinic ACh antagonist, was used, indicating that the involvement of the ACh system in this matter was indirect. The concentration of scopolamine needed for the half-maximum effect was 172-fold higher than that of propranolol. We thus conclude that the involvement of the muscarinic ACh system in ocular dominance plasticity is secondary to that of the NA-beta-adrenoreceptor system.     

Effect of neurotensin on contractile activity and [3H]acetylcholine release in cat terminal ileum during different postnatal periods.

The effect of neurotensin (NT) on the contractile activity of circular and longitudinal strips from the terminal ileum of 15-, 30-, 60-day-old and adult cats as well as on the resting and electrically-evoked release of [3H]acetylcholine (ACh) was studied. Radioactivity was measured by liquid scintillation spectrometry and the effect of NT was evaluated by the S2/S1 ratio. In the circular muscle strips NT (1-100 nM) inhibited spontaneous contractions in all age groups. In the longitudinal strips the effect of NT was concentration- and age-dependent. NT at a concentration of 1 nM had no effect on the spontaneous activity in 15-day-old cats, but in the other age groups in 70-80% of the cats it inhibited spontaneous contractions. The response to 10 and 100 nM NT was either biphasic (relaxation followed by contraction) or inhibitory: in 15-day-old cats the response was biphasic only and with increasing age the percentage of strips responding with inhibition of the contractions increased. Neither substances affecting adrenergic and cholinergic transmission nor TTX changed the inhibitory response to NT. The contractile component of the biphasic response was TTX-resistant in all age groups and was significantly decreased by scopolamine in 60-day-old and adult cats. NT increased both resting and electrically-evoked release of [3H]ACh which was not changed by TTX. In the presence of the peptide the S2/S1 ratio increased as NT-induced [3H]ACh release in the strips of adult cats was higher than that in young cats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid eye movement sleep induction by vasoactive intestinal peptide infused into the oral pontine tegmentum of the rat may involve muscarinic receptors

In rats, rapid eye movement sleep can be induced by microinjection of either the cholinergic agonist carbachol or the neuropeptide vasoactive intestinal peptide into the oral pontine reticular nucleus. Possible involvement of cholinergic mechanisms in the effect of vasoactive intestinal peptide was investigated using muscarinic receptor ligands. Sleep-waking cycles were analysed after infusion into the oral pontine reticular nucleus of vasoactive intestinal peptide (10 ng in 0.1 microl), carbachol (20 ng), atropine (200 ng) and pirenzepine (50, 100 ng), performed separately or in combination at 15-min intervals. The increase in rapid eye movement sleep due to the combined infusion of vasoactive intestinal peptide and carbachol (+58.7+/-4.6% for 8 h, P<0.05) was not significantly different from that induced by each compound separately. The enhancement of rapid eye movement sleep by vasoactive intestinal peptide was totally prevented by infusion of atropine, but not pirenzepine, a relatively selective M1 antagonist. On their own, none of the latter two compounds affected the sleep-waking cycle. Quantitative autoradiographic studies using [3H]quinuclidinyl benzylate (1 nM) and pirenzepine (0.5 microM) indicated that muscarinic receptors correspond to pirenzepine-insensitive binding sites in the oral pontine reticular nucleus. In vitro, vasoactive intestinal peptide (1-100 nM) significantly increased (+30-40%) the specific binding of [3H]quinuclidinyl benzylate to the oral pontine reticular nucleus in rat brain sections. This effect appeared to be due to an increased density, with no change in affinity, of pirenzepine-insensitive binding sites in this area. These data suggest that pirenzepine-insensitive muscarinic binding sites are involved in the induction of rapid eye movement sleep by vasoactive intestinal peptide at the pontine level in the rat.     

Picric acid-evoked release of [14C]acetylcholine from the isolated synaptosome of rat cerebral cortex

Picric acid stimulated, in a dose-dependent manner, the release of [14C]acetylcholine (ACh) from isolated synaptosomes of rat cerebral cortex pre-loaded with labelled choline. Radioactive ACh was separated for counting from choline in the synaptosomal supernatants by a liquid cation-exchange method. Neither the nicotinic antagonist (hexamethonium) nor the muscarinic antagonists (atropine and scopolamine) affected the effectiveness of picric acid, suggesting that the action of picric acid does not occur through a cholinoceptor-mediated mechanism. Moreover, oxotremorine, but not pilocarpine, inhibited ACh release in a concentration-dependent manner in either basal- or picric acid-evoked conditions, indicating the presence of muscarinic M2-receptors for auto-regulation of ACh release. The effect of picric acid was compared with high-K+ depolarization which also initiated a non-receptor-mediated release of ACh. Deletion of calcium ion from the medium negated the effects of both drugs. The ACh-releasing effect of picric acid was totally abolished, whereas high-K+ depolarization was reduced to some extent, when tetrodotoxin was added to the medium. These results indicate that picric acid acts as a releaser of ACh in the cerebrocortex of rat.     

Benzodiazepine Receptor Agonists Cause Drug-Specific and State-Specific Alterations in EEG Power and Acetylcholine Release in Rat Pontine Reticular Formation

Study Objectives:

Benzodiazepine (BDZ) and non-benzodiazepine (NBDZ) hypnotics enhance GABAergic transmission and are widely used for the treatment of insomnia. In the pontine reticular formation (PRF), GABA inhibits rapid eye movement (REM) sleep and acetylcholine (ACh) release. No previous studies have characterized the effects of BDZ and NBDZ hypnotics on ACh release in the PRF. This study tested 2 hypotheses: (1) that microdialysis delivery of zolpidem, eszopiclone, and diazepam to rat PRF alters ACh release in PRF and electroencephalographic (EEG) delta power and (2) that intravenous (IV) administration of eszopiclone to non-anesthetized rat alters ACh release in the PRF, sleep, and EEG delta power.

Design:

A within- and between-groups experimental design.

Setting:

University of Michigan.

Patients or Participants:

Adult male Crl:CD*(SD) (Sprague-Dawley) rats (n = 57).

Interventions:

In vivo microdialysis of the PRF in rats anesthetized with isoflurane was used to derive the concentration-response effects of zolpidem, eszopiclone, and diazepam on ACh release. Chronically instrumented rats were used to quantify the effects of eszopiclone (3 mg/kg, IV) on ACh release in the PRF, sleep-wake states, and cortical EEG power.

Measurements and Results:

ACh release was significantly increased by microdialysis delivery to the PRF of zolpidem and eszopiclone but not diazepam. EEG delta power was increased by zolpidem and diazepam but not by eszopiclone administered to the PRF. Eszopiclone (IV) decreased ACh release in the PRF of both anesthetized and non-anesthetized rats. Eszopiclone (IV) prevented REM sleep and increased EEG delta power.

Conclusion:

The concentration-response data provide the first functional evidence that multiple GABA_A receptor subtypes are present in rat PRF. Intravenously administered eszopiclone prevented REM sleep, decreased ACh release in the PRF, and increased EEG delta power. The effects of eszopiclone are consistent with evidence that ACh release in the PRF is lower during NREM sleep than during REM sleep, and with data showing that cholinergic stimulation of the PRF activates the cortical EEG.

Citation:

Hambrecht-Wiedbusch VS; Gauthier EA; Baghdoyan HA; Lydic R. Benzodiazepine receptor agonists cause drug-specific and state-specific alterations in EEG power and acetylcholine release in rat pontine reticular formation. SLEEP 2010;33(7):909-918.     

High spatial resolution studies of muscarinic neuroeffector junctions in mouse isolated vas deferens

It is acknowledged that neurotransmission in the mouse vas deferens is predominantly mediated by ATP and noradrenaline (NA) released from sympathetic nerves while cholinergic transmission in the rodent vas deferens is often overlooked despite early literature. Recently we have characterized a cholinergic component of neurogenic contraction of mouse isolated vas deferens. In the present paper, by confocal imaging of Ca²⁺ dynamics we detected acetylcholine (ACh) action at muscarinic cholinergic neuroeffector junctions at high-resolution. Experiments were carried out in the presence of prazosin (100 nM) and α,β methylene ATP (α,β-MeATP) (1 μM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 μM) elicited Ca²⁺ transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 μM). Ca²⁺ transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 μM). Stimulation produced a marked increase in the frequency and number of Ca²⁺ transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The α₂-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca²⁺ transients, but increased the frequency of occurrence of evoked transients, an effect inhibited by cyclopentolate. The postjunctional effects of neuronally-released ACh are limited by the action of cholinesterase. Release of ACh appears to be tonically inhibited by NA released from sympathetic nerve terminals through action at prejunctional α₂-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca²⁺ events, with no effect on Ca²⁺ transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca²⁺ transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M₃ muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca²⁺ dynamics in this tissue.     

Coupling of presynaptic muscarinic autoreceptors to serine kinases in low and high release conditions on the rat motor nerve terminal

We used intracellular recording to investigate how muscarinic acetylcholine receptors and the serine kinase signal transduction cascade are involved in regulating transmitter release in the neuromuscular synapses of the levator auris longus muscle from adult rats. Experiments with M1 and M2 selective blockers show that these subtypes of muscarinic receptors were involved in enhancing and inhibiting acetylcholine (ACh) release, respectively. Because the unselective muscarinic blocker atropine considerably increased release, the overall presynaptic muscarinic mechanism seemed to moderate ACh secretion in normal conditions. This muscarinic function did not change when more ACh was released (high external Ca2+) or when there was more ACh in the cleft (fasciculin II). However, when release was low (high external Mg2+ or low external Ca2+) or when there was less ACh in the cleft (when acetylcholinesterase was added, AChE), the response of M1 and M2 receptors to endogenously released ACh shifted to optimize release, thus producing a net potentiation of the Mg2+-depressed level. Protein kinase A (PKA) (but not protein kinase C, PKC) has a constitutive role in promoting a component of normal release because when it is inhibited with N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2 HCl, release diminishes. The imbalance of the muscarinic acetylcholine receptors (mAChRs) (with the selective block of M1 or M2) inverts the kinase function. PKC can then tonically stimulate transmitter release, whereas PKA is uncoupled. The muscarinic function can be explained by an increased M1-mediated PKC activity-dependent release and a decreased M2-mediated PKA activity-dependent release. In the presence of high external Mg2+ or low Ca2+, or when AChE is added, both mAChRs may potentiate release through an M2-mediated PKC mechanism and an M1-mediated mechanism downstream of the PKC.     

Cholinergic modulation of the locomotor network in the lamprey spinal cord

Acetylcholine (ACh) was found here to be a strong modulator of swimming activity in the isolated spinal cord preparation of the adult lamprey (Ichthyomyzon unicuspis). During fictive swimming induced with either D-glutamate or N-methyl-D-aspartate, addition of ACh (200 microM) significantly reduced the cycle period of ventral root bursts to 54%, intersegmental phase lag to 32%, and ventral root burst proportion to 80% of control levels. Effects of ACh were apparent at concentrations as low as 1 microM. Both nicotinic and muscarinic receptors are involved, in that application of either nicotinic or muscarinic agonists alone significantly reduced cycle period. There is sufficient endogenous ACh in the spinal cord to modulate ongoing fictive swimming, as shown by application of the cholinesterase inhibitor eserine (physostigmine). Eserine (20 microM) significantly reduced the cycle period to 78% and phase lag to 58% of control levels, and these effects were reversed with the addition of cholinergic blockers. Addition of only a nicotinic or muscarinic antagonist, mecamylamine (10 microM) or scopolamine (20 microM), respectively, to the spinal cord during fictive swimming produced significant increases in cycle period and phase lag, suggesting that both types of cholinergic receptors participate in endogenous cholinergic modulation. It is concluded that ACh is an endogenous modulator of the locomotor network in the lamprey spinal cord and that ACh may take part in the regulation of cycle period, intersegmental coupling, and ventral root burst duration.