The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype.

Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a down-regulation in the expression of ATP "binding cassette" G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors.

Carbachol alleviates myocardial injury in septic rats through PI3K/AKT signaling pathway.

OBJECTIVE: To explore the effect of carbachol on myocardial injury in septic rats, and to further study its influence on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. MATERIALS AND METHODS: A total of 48 healthy male Sprague-Dawley rats were randomly divided into sham group (n=16), model group (n=16), and carbachol group (n=16). The rat model of sepsis was established via cecal ligation and puncture. Carbachol was intraperitoneally injected (10 µg/kg) immediately after operation in carbachol group, and no cecal ligation was performed in sham group. At 48 h after operation, the survival rate of rats in each group was recorded, the activity of plasma creatine kinase-MB (CK-MB) was detected, and the cardiac function in each group was determined. Moreover, the heart was isolated, and the myocardial tissues were taken to detect the apoptosis level using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis kit. The content of inflammatory factors in myocardial tissues was determined using enzyme-linked immunosorbent assay (ELISA) kits, and the expression levels of apoptosis-related proteins and the PI3K/AKT signaling pathway-related proteins were detected via Western blotting. RESULTS: Carbachol could significantly raise the survival rate of septic rats (p<0.01), remarkably decrease the activity of CK-MB (p<0.01), markedly reduce the left ventricular internal diameter at end-systole (LVIDs), and markedly increase the left ventricular ejection fraction (LVEF, %) and left ventricular fractional shortening (LVFS, %). Besides, carbachol could evidently lower the apoptosis level of myocardial cells of septic rats (p<0.01), reduce the content of inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 (p<0.01), notably decrease the expression of Caspase-3 in myocardial tissues (p<0.01), remarkably increase the expression of Bcl-2/Bax (p<0.01), and distinctly inhibit the expressions of phosphorylated (p-)PI3K, p-AKT, Nod-like receptor protein 3 (NLRP3), and Caspase-1 (p<0.01). CONCLUSIONS: Carbachol can reduce the release of inflammatory factors in myocardial cells, the expression of apoptotic proteins and the apoptosis of myocardial cells, and improve the cardiac function and survival rate of septic rats by inhibiting the PI3K/AKT signaling pathway.

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

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

Adaptive short-term associative conditioning in the pancreatic ß-cell.

This study associates cholinergic stimulation of the pancreatic ß-cell electrical activity with a short-term memory phenomenon. Glucose pulses applied to a basal glucose concentration induce depolarizing waves which are used to estimate the evolution of the ß-cell glucose sensitivity. Exposure to carbamoylcholine (carbachol) increases the size of the glucose-induced depolarizing waves. This change appears after carbachol withdrawal and implies a temporal potentiation of sensitivity (TPS) lasting up to one hour. TPS induction requires the simultaneous action of carbachol and glucose. The substitution of glucose with the secretagogues glyceraldehyde or 2-ketoisocaproate mimics glucose-induced TPS, while palmitate does not. TPS is not produced if the membrane is kept hyperpolarized by diazoxide. Glucose can be replaced by tolbutamide, suggesting a role of depolarization and a subsequent increase in intracellular calcium concentration. A role for kinases is suggested because staurosporine prevents TPS induction. Cycloheximide does not impair TPS induction, indicating that de novo protein synthesis is not required. The fact that the two inputs acting simultaneously produce an effect that lasts up to one hour without requiring de novo protein synthesis suggests that TPS constitutes a case of short-term associative conditioning in non-neural tissue. The convergence of basal glucose levels and muscarinic activation happens physiologically during the cephalic phase of digestion, in order to later absorb incoming fuels. Our data reveals that the role of the cephalic phase may be extended, increasing nutrient sensitivity during meals while remaining low between them.

Effect of low dose metronomic therapy on MCF-7 tumor cells growth and angiogenesis. Role of muscarinic acetylcholine receptors.

The non-neuronal cholinergic system refers to the presence of acetylcholine, choline acetyltransferase, acetylcholinesterase and cholinergic receptors, nicotinic and muscarinic (mAChRs) expressed in non-neuronal cells. The presence of mAChRs has been detected in different type of tumor cells and they are linked with tumorigenesis. We had previously documented the expression of mAChRs in murine and human mammary adenocarcinomas and the absence of these receptors in normal mammary cells of the same origins. We also demonstrated that mAChRs are involved in breast cancer progression, pointing to a main role for mAChRs as oncogenic proteins. Since the long term treatment of breast cancer cells with the muscarinic agonist carbachol promoted cell death, here we investigated the ability of low doses of this agonist combined with paclitaxel (PX), a taxane usually administered to treat breast cancer, to inhibit the progression of human MCF-7 tumor cells. We demonstrated that PX plus carbachol reduced cell viability and tumor growth in vitro probably due to a down-regulation in cancer stem cells population and in the expression of ATP "binding cassette" G2 drug extrusion pump; also a reduction in malignant-induced angiogenesis was produced by the in vivo administration of the mentioned combination in a metronomic schedule to MCF-7 tumor-bearing NUDE mice. Our results confirm that mAChRs could be considered as therapeutic targets for metronomic therapy in breast cancer as well as the usefulness of a muscarinic agonist as repositioning drug in the treatment of this type of tumors.

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

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

Dissimilar interaction between dopaminergic and cholinergic systems in the initiation of emission of 50-kHz and 22-kHz vocalizations.

Rats emit 22-kHz or 50-kHz ultrasonic vocalizations (USVs) to signal their emotional state to other conspecifics. The 22-kHz USVs signal a negative emotional state while 50-kHz USVs reflect a positive affective state. The initiation of 22-kHz USVs is dependent on the activity of cholinergic neurons within the laterodorsal tegmental nucleus that release acetylcholine along the medial cholinoceptive vocalization strip. Emission of 50-kHz USVs is dependent upon the activation of dopaminergic neurons located within the ventral tegmental area that release dopamine into the medial shell of the nucleus accumbens. There have been reports that showed an antagonistic interaction between acetylcholine and dopamine during the expression of emotional states, and dopamine agonists decreased carbachol-induced emission of 22-kHz USVs. The current study tests the hypothesis that initial antagonism of dopamine receptors by systemic haloperidol or intraacumbens raclopride should increase the subsequent emission of 22 kHz USVs induced by carbachol from the lateral septum. Our findings showed that antagonism of dopaminergic signaling either via systemic haloperidol or via intracerebral raclopride did not alter the number of emitted 22-kHz USVs. Thus, inhibition of the mesolimbic dopamine system did not increase the magnitude of a negative emotional state. It was found, however, that prolonged emission of 22-kHz USVs initiated by carbachol caused a delayed rebound emission (R) of 50-kHz USVs appearing after 300 s of emission of 22-kHz USVs, i.e., when the response was subsiding. The R-50-kHz USVs were predominantly frequency modulated (FM) USVs and their number was directly proportional to the number of recorded 22-kHz USVs. The emission of R-50-kHz USVs was reversed by systemic pretreatment with haloperidol or intraacumbens injection of raclopride. It is argued that the R-50-kHz USVs represent a rebound emotional state that is opposite in valence and arousal induced by carbachol. Importantly, prolonged emission of amphetamine-induced 50 kHz USVs failed to show any vocalization rebound effect.

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

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

The antagonistic relationship between aversive and appetitive emotional states in rats as studied by pharmacologically-induced ultrasonic vocalization from the nucleus accumbens and lateral septum.

Rats can emit 22-kHz or 50-kHz ultrasonic vocalizations (USVs) in negative, as well as positive contexts which index their emotional state. 22-kHz USVs are emitted during aversive contexts and can be initiated by activation of the ascending cholinergic pathways originating from the laterodorsal tegmental nucleus or initiated pharmacologically by injection of cholinergic agonists into target areas of these pathways (medial cholinoceptive vocalization strip). Conversely, 50-kHz USVs are emitted during positive pro-social contexts and can be initiated by stimulation of ascending dopaminergic pathways originating from the ventral tegmental area or by injection of dopamine agonists into target areas of these pathways (nucleus accumbens shell). Recently, we have shown an inhibitory effect a positive emotional state has on the emission of carbachol-induced 22-kHz USVs from the anterior hypothalamic/medial preoptic area. However, this structure is a fragment of that cholinoceptive vocalization strip. We wanted to examine if we could observe similar effect when the aversive state is induced from the lateral septum, the most rostral division of the cholinoceptive vocalization strip. The results supported previous findings. First, microinjection of the dopamine agonist R-(-)-apomorphine into the nucleus accumbens shell resulted in increased emission of frequency modulated (FM) 50-kHz USVs that are regarded as signals expressing a positive emotional state in rats. Second, FM 50-kHz USVs and not flat (F) 50-kHz USVs were able to decrease 22-kHz USVs induced by microinjections of carbachol into the lateral septum. This research provides further support to the hypothesis that the initiation of a positive emotional state functionally antagonizes initiation of a negative emotional state in rats.

NMDA Receptor-Dependent Cholinergic Modulation of Mesolimbic Dopamine Cell Bodies: Neurochemical and Behavioral Studies.

Substance abuse disorders are devastating, costly, and difficult to treat. Identifying the neurochemical mechanisms underlying reinforcement promises to provide critical information in the development of effective treatments. Several lines of evidence suggest that striatal dopamine (DA) release serves as a teaching signal in reinforcement learning, and that shifts in DA release from the primary reward to reward-predicting stimuli play a critical role in the self-administration of both natural and non-natural rewards. However, far less is known about the reinforcing effects of motivationally neutral sensory stimuli, or how these signals can facilitate self-administration behavior. Thus, we trained rats ( n = 7) to perform a visual stimulus-induced instrumental task, which involved lever pressing for activation of a stimulus light. We then microinfused vehicle (phosphate buffered saline), carbachol (acetylcholine receptor agonist), or carbachol in the presence of an N-methyl-d-aspartate (NMDA) receptor-specific drug (NMDA itself, or the antagonist, AP5) into the ventral tegmental area (VTA). This enabled us to directly evaluate how chemical modulation of dopamine cell bodies affects the instrumental behavior, as well as the nature of extracellular dopamine transients recorded in the nucleus accumbens shell (NAc shell) using fast-scan cyclic voltammetry (FSCV). Intra-VTA infusion of carbachol enhanced the magnitude and frequency of dopamine transients in the NAc shell and potentiated active lever responding without altering inactive lever responding, as compared to infusion of vehicle. Coinfusion of carbachol with AP5 abolished dopamine transients recorded in the NAc and attenuated active lever responding without altering inactive lever responding. Finally, coadministration of carbachol and NMDA into the VTA restored both lever pressing and dopaminergic signals recorded in the striatum. Together, these results suggest that acetylcholine and glutamate synergistically act at dopamine cells in the VTA to modulate VTA-NAc shell dopaminergic output, and this underlies motivation to lever press for a motivationally neutral visual stimulus.

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

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

Endogenous hydrogen peroxide affects antidiuresis to cholinergic activation in the medial septal area.

Cholinergic activation of the medial septal area (MSA) with carbachol produces thirst, natriuresis and antidiuresis. Hydrogen peroxide (H2O2) injected into the medial septal area (MSA) impairs behavioral and renal responses induced by carbachol at the same site, suggesting the exogenous H2O2 may modulate the responses to cholinergic activation in the MSA. In the present study, we investigated if the accumulation of endogenous H2O2 in the MSA after the injection of the catalase inhibitor 3-amino-1,2,4-triazole (ATZ) also affects cholinergic responses. In addition, the effects of the combination of ATZ with a non-effective dose of H2O2 in the MSA were also tested. Male Holtzman rats (280-320 g) with stainless steel cannulas implanted in the MSA were used. The treatment with ATZ (10 nmol) into the MSA partially reverted the antidiuretic effect of carbachol (10.5 ± 0.7, vs. saline + carbachol: 7.3 ± 0.6 ml/120 min), without changing carbachol-induced water intake (9.5 ± 1.9, vs. saline + carbachol: 10.7 ± 1.6 ml/60 min). The combination of a low dose of ATZ (2.5 nmol) with an ineffective dose of H2O2 (0.5 µmol) into the MSA reduced carbachol-induced thirst (7.5 ± 2.0, vs. saline + carbachol: 14.9 ± 1.2 ml/15 min) and reverted the antidiuresis (8.1 ± 1.1, vs. saline + carbachol: 5.3 ± 0.9 ml/120 min). Sodium and potassium excretion were not modified regardless the treatment. Although exogenous H2O2 injected in the MSA may affect most of the responses to cholinergic activation of the MSA, the antidiuresis is the response clearly modulated by endogenous H2O2.

Cholinergic responses of acoustically-characterized cochlear nucleus neurons: An in vivo iontophoretic study in Guinea pig.

The responses of guinea pig cochlear nucleus neurons to in vivo iontophoretic application of various neurotransmitter agonists were recorded with extracellular multi-barrelled electrodes. Where possible, neurons were physiologically identified using strict criteria. Emphasis was placed on the action of cholinergic agonists in relation to the possible action of olivocochlear collateral innervation. Excitatory responses (increase in action potential firing) to glutamate were confirmed in a number of neuronal response types. Application of acetylcholine (ACh) or the broad spectrum cholinergic agonist carbachol produced reliable excitatory responses in about 47% of neurons (n = 29 out of 61 neurons). The remaining neurons were unresponsive to cholinergic agonists and no inhibitory responses were observed. Cholinergic responses were more common in dorsal cochlear nucleus (DCN) (73% of 30 neurons tested) than in ventral cochlear nucleus (VCN) (23% of 31 neurons). Of the total neuron sample in which cholinergic responses were investigated, 41 neurons were able to be categorized according to established acoustic response features. Excitatory responses to cholinergic agonists were seen in "Pauser-buildup" (Pb) and "Transient chopper" (Ct) response types. Primary-like neurons (PL and Pn) as well as "Onset chopper" (Oc) neurons (n = 6) were unresponsive to either ACh or carbachol. Oc neurons also did not show any effect on their acoustic responses. Robust cholinergic responses were also seen in several VCN and DCN neurons that were either unresponsive to sound, or had acoustic response properties that did not fit standard classification. The results suggest a relatively more robust cholinergic innervation of DCN compared to VCN. The excitatory cholinergic responses of some Ct neurons and the lack of effect on Oc neurons are consistent with previous results in mouse brain slice studies, but are in conflict with reports of medial olivocochlear collateral excitatory responses in onset-type neurons in vivo. The results also indicate that a number of neurons of unknown identity may also receive cholinergic input.

Are There Pharmacotherapeutic Options for Underactive Bladder?

Currently, underactive bladder, the symptom-based correlate of the urodynamic diagnosis of detrusor underactivity, has no effective drug treatments. Use of sympathomimetics, targeting cholinergic receptors, is not supported by current evidence. Several potential targets are the subject of ongoing investigation.

Precisely Timed Nicotinic Activation Drives SST Inhibition in Neocortical Circuits.

Sleep, waking, locomotion, and attention are associated with cell-type-specific changes in neocortical activity. The effect of brain state on circuit output requires understanding of how neuromodulators influence specific neuronal classes and their synapses, with normal patterns of neuromodulator release from endogenous sources. We investigated the state-dependent modulation of a ubiquitous feedforward inhibitory motif in mouse sensory cortex, local pyramidal (Pyr) inputs onto somatostatin (SST)-expressing interneurons. Paired whole-cell recordings in acute brain slices and in vivo showed that Pyr-to-SST synapses are remarkably weak, with failure rates approaching 80%. Pharmacological screening revealed that cholinergic agonists uniquely enhance synaptic efficacy. Brief, optogenetically gated acetylcholine release dramatically enhanced Pyr-to-SST input, via nicotinic receptors and presynaptic PKA signaling. Importantly, endogenous acetylcholine release preferentially activated nicotinic, not muscarinic, receptors, thus differentiating drug effects from endogenous neurotransmission. Brain state- and synapse-specific unmasking of synapses may be a powerful way to functionally rewire cortical circuits dependent on behavioral demands.

Existence of nicotinic receptors in a subset of type I vestibular hair cells of guinea pigs.

In mammals, vestibular hair cells (VHCs) are classified as type I and II according to morphological criteria. Acetylcholine (ACh) is identified as the primary efferent neurotransmitter. To date, cholinergic activities have been reported in mammalian type II VHCs, but similar activities in type I VHCs have not been pursued presumably because the body of type I VHCs were suggested to be totally surrounded by afferent nerve calyces. A few reports showed that part of type I VHCs were incompletely surrounded by calyces and received contact from the efferent nerve endings in the mammals studied. The possibility of the expression of cholinergic receptors, their subunit composition, and their function in mammals' type I VHCs are still unclear. In this study, nicotinic responses were investigated by the whole-cell patch clamp technique in isolated type I VHCs of guinea pigs. Of the cells, 7.3% were sensitive to cholinergic agonists and showed an excitatory current at -40 mV which was not sensitive to nifedipine, iberiotoxin (IBTX), and apamin. The main carriers of this current were Na+ and K+. The rank order of activation potency was nicotine > 1,1-dimethyl-4-phenyl-piperazinium (DMPP) > ACh. These nicotinic ACh receptors (nAChRs) were not blocked by strychnine and α-bungarotoxin (α-BTX), but sensitive to d-tubocurarine (dTC) and mecamylamine (Mec). The findings provide physiological evidence that some subtypes of nAChRs may be located in a subset of type I VHCs, which were different from α9α10 nAChRs.

Effect of constant light and immobilization stress on rat submandibular saliva secretory response induced by cholinergic and peptidergic agonists

The aim of this work was to analyse the parasympathetic control of submandibular saliva secretory response to cholinergic and peptidergic agonists in rats chronically exposed to constant light or repeated immobilization. Thirty two adult male Wistar rats were used: LL (8 rats exposed to constant light for 20 days), IMO (8 rats submitted to 14:10 h light: dark cycle and immobilized 2 hours daily for 7 days), and control (16 rats not exposed to stress and submitted to 14:10 hours light:dark cycle). Saliva was collected under anesthesia from the salivary ducts of submandibular glands under increasing doses of methacholine and substance P. Secretory responses (µg/saliva/mg dry weight gland) to methacholine were significantly higher in LL and IMO groups compared to control for the following doses (µg/kg body weight): 3 (153±9 versus 46±3, p<0.001 and 76±3 versus 40±3, p<0.001), 10 (379±23 versus 277±8, p<0.001 and 275±19 versus 250±10, p<0.01) and 30 (729±25 versus 695±19, p<0.05 and 1008±39 versus 640±20, p<0.001). Also, responses to substance P were significantly increased in LL and IMO groups compared to control for the following doses: 0.2 (80±3 versus 30±3, p<0.01 and 94±16 versus 31±3, p<0.001), 0.5 (328±20 versus 231±16, p<0.01 and 531±31 versus 219±25,p<0.001), 1 (681±35 versus 547±30, p<0.01 and 1031±63 versus 563±53, p<0.001), and 5 (2222±88 versus 1868±59, p<0.01 and 3230±145 versus 1921±218, p<0.001). In conclusion, supersensitivity of secretory response to both agonists suggests that chronic exposure of rats to stressors capable of activating the sympathetic adrenal system promotes inhibition of the parasympathetic control of salivary secretion.

Modulation of macrophage phagocytosis in vitro-A role for cholinergic stimulation?

Acetylcholine is synthetized and released from neural cells, but also by non-neuronal cells such as epithelial cells or keratinocytes. Cholinergic agonists enhance the phagocytosis of zymosan particles in primary peritoneal macrophages. The aim of this study was to investigate the effect of carbachol stimulation on phagocytosis in a macrophage cell line using microspheres. The murine cell line MH-S was used in a phagocytosis assay with fluorescent latex beads. The amount of the ingested beads was determined using flow cytometry. Gene expression was investigated using polymerase chain reaction. Gene expression of the muscarinic receptors M1, M3, M4 and M5 but not M2 was found. Carbachol slightly increased the phagocytosis of microspheres in the macrophages. A co-stimulation using lipopolysaccharide and carbachol did not increase the effect of lipopolysaccharide alone. In conclusion, cholinergic stimulation in vitro only moderately modulates the phagocytosis of microspheres. M2 might have a role in stimulation of macrophage phagocytosis.

Increase in sensitivity of the baroreceptor reflex following microinjection of carbachol into the posterior hypothalamic nucleus of awake rats.

In a rat model, the baroreceptor reflex can be assessed by graded infusions of either phenylephrine or sodium nitroprusside with continuous hemodynamic monitoring. Microinjection of the cholinergic agonist carbachol (CCh) into the posterior hypothalamic nucleus (PHN) evokes an increase in mean arterial pressure and a change in heart rate. Lower doses of CCh evoke only tachycardia, whereas middle and higher doses evoke a biphasic change in heart rate of tachycardia followed by bradycardia. The bradycardia following the microinjection of CCh into the PHN can be attenuated by the previous administration of the vasopressin V1 receptor antagonist [d(CH2 )5 Tyr(Me)] arginine vasopressin (AVPX). Circulating arginine vasopressin (AVP) has been shown to increase the sensitivity of the baroreceptor reflex by stimulating vasopressin V1 receptors in the area postrema. The attenuation by AVPX of the bradycardia that results following the high doses of CCh suggests that AVP is released into the circulation following stimulation of cholinergic systems within the PHN. Thus, microinjection of a high dose of CCh (11 nmol) into the PHN alters the sensitivity of the baroreceptor reflex by increasing peripheral levels of AVP.

Cell discharge correlates of posterior hypothalamic theta rhythm. Recipe for success in recording stable field potential.

The theta rhythm discovered in the posterior hypothalamus area (PHa) differs from theta observed in the hippocampal formation. In comparison to hippocampal spontaneous theta, the theta recorded in the PHa is rarely registered, has lower amplitude, often disappears, and sometimes returns after a few minutes. These features indicate that spontaneous theta recorded in the PHa is not an appropriate experimental model to search for the correlation between PHa cell discharges and local field potential. In this paper we present standard experimental conditions necessary to record theta-related cells in the PHa in anesthetized rats. Three pharmacological agents were used in the experiments to induce PHa theta rhythm in urethanized rats: carbachol (CCH), carbenoxolone and kainic acid, which are potent enough to induce well-synchronized PHa theta. However, CCH was found to be the best pharmacological tool to induce PHa theta oscillations, due to its longest duration of action and lack of preliminary epileptogenic effects. It seems that CCH-induced theta can be the most suitable pharmacological model for experiments with the use of protocol of long-lasting recordings of PHa theta-related cell discharges.