Regulating nociceptive transmission by VGluT2-expressing spinal dorsal horn neurons.

Vesicular glutamate transporter-2 (VGluT2) mediates the uptake of glutamate into synaptic vesicles in neurons. Spinal cord dorsal horn interneurons are highly heterogeneous and molecularly diverse. The functional significance of VGluT2-expressing dorsal horn neurons in physiological and pathological pain conditions has not been explicitly demonstrated. Designer receptors exclusively activated by designer drugs (DREADDs) are a powerful chemogenetic tool to reversibly control neuronal excitability and behavior. Here, we used transgenic mice with Cre recombinase expression driven by the VGluT2 promoter, combined with the chemogenetic approach, to determine the contribution of VGluT2-expressing dorsal horn neurons to nociceptive regulation. Adeno-associated viral vectors expressing double-floxed Cre-dependent Gαq-coupled human M3 muscarinic receptor DREADD (hM3D)-mCherry or Gαi-coupled κ-opioid receptor DREADD (KORD)-IRES-mCitrine were microinjected into the superficial spinal dorsal horn of VGluT2-Cre mice. Immunofluorescence labeling showed that VGluT2 was predominantly expressed in lamina II excitatory interneurons. Activation of excitatory hM3D in VGluT2-expressing neurons with clozapine N-oxide caused a profound increase in neuronal firing and synaptic glutamate release. Conversely, activation of inhibitory KORD in VGluT2-expressing neurons with salvinorin B markedly inhibited neuronal activity and synaptic glutamate release. In addition, chemogenetic stimulation of VGluT2-expressing neurons increased mechanical and thermal sensitivities in naive mice, whereas chemogenetic silencing of VGluT2-expressing neurons reversed pain hypersensitivity induced by tissue inflammation and peripheral nerve injury. These findings indicate that VGluT2-expressing excitatory neurons play a crucial role in mediating nociceptive transmission in the spinal dorsal horn. Targeting glutamatergic dorsal horn neurons with inhibitory DREADDs may be a new strategy for treating inflammatory pain and neuropathic pain.

Inter- and intra-specific differences in muscarinic acetylcholine receptor expression in the neural pathways for vocal learning in songbirds.

Acetylcholine receptors (AChRs) abound in the central nervous system of vertebrates. Muscarinic AChRs (mAChRs), a functional subclass of AChRs, mediate neuronal responses via intracellular signal transduction. They also play roles in sensorimotor coordination and motor skill learning by enhancing cortical plasticity. Learned birdsong is a complex motor skill acquired through sensorimotor coordination during a critical period. However, the functions of AChRs in the neural circuits for vocal learning and production remain largely unexplored. Here, we report the unique expression of mAChRs subunits (chrm2-5) in the song nuclei of zebra finches. The expression of excitatory subunits (chrm3 and chrm5) was downregulated in the song nuclei compared with the surrounding brain regions. In contrast, the expression of inhibitory mAChRs (chrm2 and chrm4) was upregulated in the premotor song nucleus HVC relative to the surrounding nidopallium. Chrm4 showed developmentally different expression in HVC during the critical period. Compared with chrm4, individual differences in chrm2 expression emerged in HVC early in the critical period. These individual differences in chrm2 expression persisted despite testosterone administration or auditory deprivation, which altered the timing of song stabilization. Instead, the variability in chrm2 expression in HVC correlated with parental genetics. In addition, chrm2 expression in HVC exhibited species differences and individual variability among songbird species. These results suggest that mAChRs play an underappreciated role in the development of species and individual differences in song patterns by modulating the excitability of HVC neurons, providing a potential insight into the gating of auditory responses in HVC neurons.

Spinalization and locomotor training differentially affect muscarinic acetylcholine receptor type 2 abutting on α-motoneurons innervating the ankle extensor and flexor muscles.

Complete thoracic spinal cord transection (SCT) impairs excitatory cholinergic inputs to ankle extensor (soleus; Sol) but not to flexor (tibialis anterior; TA) α-motoneurons (MNs) modifiable by locomotor training applied post-transection. The purpose of this study was to investigate whether Sol and TA MNs adapt to changes in cholinergic environment by differential regulation of their muscarinic receptors M2 (M2R). We examined Chrm2 (M2R gene) transcript level, high-affinity 3-quinuclidinyl benzilate-3 H ([3 H]QNB) ligand binding, distribution and density of M2R immunolabeling in lumbar (L) segments in intact and SCT rats, with or without inclusion of 5-week treadmill locomotor training. We show that at the second week after SCT the levels of Chrm2 transcript are reduced in the L3-6 segments, with [3 H]QNB binding decreased selectively in the L5-6 segments, where ankle extensor MNs are predominantly located. At 5 weeks after SCT, [3 H]QNB binding differences between the L3-4 and L5-6 segments are maintained, accompanied by higher density of M2R immunolabeling in the plasma membrane and cytoplasm of TA than Sol MNs and by enriched synaptic versus extrasynaptic M2R pools (52% TA vs. 25% Sol MNs). Training normalized M2R in TA MNs, improved locomotion, and reduced frequency of clonic episodes. Our findings indicate higher sensitivity of TA than Sol MNs to cholinergic signaling after SCT, which might shorten flexor twitches duration and contribute to generation of clonic movements. Synaptic enrichment in M2R density may reflect a compensatory mechanism activated in TA and Sol MNs to different extent in response to reduced strength of cholinergic signaling to each MN pool. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback.

We have recently identified a cholinergic chemosensory cell in the urethral epithelium, urethral brush cell (UBC), that, upon stimulation with bitter or bacterial substances, initiates a reflex detrusor activation. Here, we elucidated cholinergic mechanisms that modulate UBC responsiveness. We analyzed muscarinic acetylcholine receptor (M1-5 mAChR) expression by using RT-PCR in UBCs, recorded [Ca2+]i responses to a bitter stimulus in isolated UBCs of wild-type and mAChR-deficient mice, and performed cystometry in all involved strains. The bitter response of UBCs was enhanced by global cholinergic and selective M2 inhibition, diminished by positive allosteric modulation of M5, and unaffected by M1, M3, and M4 mAChR inhibitors. This effect was not observed in M2 and M5 mAChR-deficient mice. In cystometry, M5 mAChR-deficient mice demonstrated signs of detrusor overactivity. In conclusion, M2 and M5 mAChRs attenuate the bitter response of UBC via a cholinergic negative autocrine feedback mechanism. Cystometry suggests that dysfunction, particularly of the M5 receptor, may lead to such symptoms as bladder overactivity.-Deckmann, K., Rafiq, A., Erdmann, C., Illig, C., Durschnabel, M., Wess, J., Weidner, W., Bschleipfer, T., Kummer, W. Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback.

Promoter IV-BDNF deficiency disturbs cholinergic gene expression of CHRNA5, CHRM2, and CHRM5: effects of drug and environmental treatments.

Brain-derived neurotrophic factor (BDNF) promotes maturation of cholinergic neurons. However, how activity-dependent BDNF expression affects specific cholinergic gene expression remains unclear. This study addressed this question by determining mRNA levels of 22 acetylcholine receptor subunits, the choline transporter (CHT), and the choline acetyltransferase (ChAT) in mice deficient in activity-dependent BDNF via promoter IV (KIV) and control wild-type mice. Quantitative RT-PCR revealed significant reductions in nicotinic acetylcholine receptor alpha 5 (CHRNA5) in the frontal cortex and hippocampus and M5 muscarinic acetylcholine receptor (CHRM5) in the hippocampus, but significant increases in M2 muscarinic acetylcholine receptor (CHRM2) in the frontal cortex of KIV mice compared to wild-type mice. Three-week treatments with fluoxetine, phenelzine, duloxetine, imipramine, or an enriched environment treatment (EET) did not affect the altered expression of these genes except that EET increased CHRNA5 levels only in KIV frontal cortex. EET also increased levels of CHRNA7, CHT, and ChAT, again only in the KIV frontal cortex. The imipramine treatment was most prominent among the four antidepressants; it up-regulated hippocampal CHRM2 and frontal cortex CHRM5 in both genotypes, and frontal cortex CHRNA7 only in KIV mice. To the best of our knowledge, this is the first evidence that BDNF deficiency disturbs expression of CHRNA5, CHRM2, and CHRM5. Our results suggest that promoter IV-BDNF deficiency - which occurs under chronic stress - causes cholinergic dysfunctions via these receptors. EET is effective on CHRNA5, while its compensatory induction of other cholinergic genes or drugs targeting CHRNA5, CHRM2, and CHRM5 may become an alternative strategy to reverse these BDNF-linked cholinergic dysfunctions.

Effect of umbilical cord blood stem cells transplantation on bladder dysfunction induced by cerebral ischemia in rats.

OBJECTIVE: To demonstrate the effect of human umbilical cord blood-derived CD34+ cells on bladder dysfunction induced by cerebral ischemia in rats. MATERIALS AND METHODS: Female rats were subjected to either 60 minute middle cerebral artery occlusion (MCAO) or a sham operation. Rats were divided into four groups: sham operation, MCAO without treatment, infusion with 1×106 CD34+ cells 30 minutes before MCAO, and infusion with 1×106 CD34+ cells 3 hours after MCAO. Bladder function was analyzed by cystometry at 1 day, 3 days, and 7 days after MCAO. Expressions of nerve growth factor (NGF), M2 and M3 muscarinic receptors were measured by immunohistochemistry and real time polymerase chain reaction. RESULTS: Cystometric results showed that, following MCAO, rats have a significant increase in peak voiding pressure and residual volume. Conversely, there is a significant decrease in voided volumes and intercontraction intervals. Cystometric variables after pre- and postischemic CD34+ treatment nearly returned to levels found in sham-operated rats. The expression of bladder NGF and M3 was decreased after MCAO, but significantly increased following preischemic CD34+ treatment. There was decreased expression of bladder M2 mRNA despite an increased level of M2 immunoreactivity at 3 days and 7 days after MCAO. Expression of bladder M2 immunoreactivity and mRNA nearly returned to sham group levels after preischemic CD34+ treatment. CONCLUSION: Bladder dysfunction in a rat model caused by MCAO may be restored to normal micturition by treatment with human umbilical cord blood-derived CD34+ cells and may be related to the expressions of NGF, M2, and M3 in the bladder.

Localization of the M2 muscarinic cholinergic receptor in dendrites, cholinergic terminals, and noncholinergic terminals in the rat basolateral amygdala: An ultrastructural analysis.

Activation of M2 muscarinic receptors (M2Rs) in the rat anterior basolateral nucleus (BLa) is critical for the consolidation of memories of emotionally arousing events. The present investigation used immunocytochemistry at the electron microscopic level to determine which structures in the BLa express M2Rs. In addition, dual localization of M2R and the vesicular acetylcholine transporter protein (VAChT), a marker for cholinergic axons, was performed to determine whether M2R is an autoreceptor in cholinergic axons innervating the BLa. M2R immunoreactivity (M2R-ir) was absent from the perikarya of pyramidal neurons, with the exception of the Golgi complex, but was dense in the proximal dendrites and axon initial segments emanating from these neurons. Most perikarya of nonpyramidal neurons were also M2R-negative. About 95% of dendritic shafts and 60% of dendritic spines were M2 immunoreactive (M2R(+) ). Some M2R(+) dendrites had spines, suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of nonpyramidal neurons. M2R-ir was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M2R(+) terminals forming asymmetrical (putative excitatory) synapses were dendritic spines, most of which were M2R(+) . The main targets of M2R(+) terminals forming symmetrical (putative inhibitory or neuromodulatory) synapses were unlabeled perikarya and M2R(+) dendritic shafts. M2R-ir was also seen in VAChT(+) cholinergic terminals, indicating a possible autoreceptor role. These findings suggest that M2R-mediated mechanisms in the BLa are very complex, involving postsynaptic effects in dendrites as well as regulating release of glutamate, γ-aminobutyric acid, and acetylcholine from presynaptic axon terminals. J. Comp. Neurol. 524:2400-2417, 2016. © 2016 Wiley Periodicals, Inc.

Urinary Retention, Incontinence, and Dysregulation of Muscarinic Receptors in Male Mice Lacking Mras.

Here we show that male, but not female mice lacking expression of the GTPase M-Ras developed urinary retention with distention of the bladder that exacerbated with age but occurred in the absence of obvious anatomical outlet obstruction. There were changes in detrusor morphology in Mras-/- males: Smooth muscle tissue, which exhibited a compact organization in WT mice, appeared disorganized and became increasingly 'layered' with age in Mras-/- males, but was not fibrotic. Bladder tissue near the apex of bladders of Mras-/- males exhibited hypercontractility in response to the cholinergic agonist carbachol in in vitro, while responses in Mras-/- females were normal. In addition, spontaneous phasic contractions of detrusors from Mras-/- males were increased, and Mras-/- males exhibited urinary incontinence. We found that expression of the muscarinic M2 and M3 receptors that mediate the cholinergic contractile stimuli of the detrusor muscle was dysregulated in both Mras-/- males and females, although only males exhibited a urinary phenotype. Elevated expression of M2R in young males lacking M-Ras and failure to upregulate M3R with age resulted in significantly lower ratios of M3R/M2R expression that correlated with the bladder abnormalities. Our data suggests that M-Ras and M3R are functionally linked and that M-Ras is an important regulator of male bladder control in mice. Our observations also support the notion that bladder control is sexually dimorphic and is regulated through mechanisms that are largely independent of acetylcholine signaling in female mice.

Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.

PURPOSE: This study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis. METHODS: Female SD rats were injected with cyclophosphamide (75 mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8 µm thickness, fixed in 70% ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R). RESULTS: The expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis. CONCLUSIONS: The LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial-afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.

Botulinum toxin A detrusor injections reduce postsynaptic muscular M2, M3, P2X2, and P2X3 receptors in children and adolescents who have neurogenic detrusor overactivity: a single-blind study.

OBJECTIVE: To analyze the effect of OnabotulinumtoxinA detrusor injections on postsynaptic muscular receptors in children and adolescents with neurogenic detrusor overactivity. MATERIALS AND METHODS: A bladder augmentation became necessary in 10 children and adolescents (7 males, 3 females; median age, 12 years) who had neurogenic detrusor overactivity. Seven had previously received 1 to 8 (average 3.86) OnabotulinumtoxinA detrusor injections, but their detrusor pressure could not be maintained at tolerable levels because of low-compliance bladder. The last injection session had been completed an average of 3 months (range, 1.5-3.5 months) previously. Three patients had never received that therapy and were considered controls. On the bladder dome resections, a specific receptor analysis (muscarinic M2 and M3 and purinergic P2X1, P2X2, and P2X3) was performed with confocal immunofluorescence, and nerve fiber density was analyzed with light-microscopic 3,3'-diaminobenzidine-immunohistochemical staining. RESULTS: Receptor analysis showed a downregulation of all examined receptors after OnabotulinumtoxinA injections; the reductions in M2, M3, P2X2, and P2X3 receptors reached a significance level of P <.05 (Mann-Whitney test). The ratios of means (OnabotulinumtoxinA-to-control) were 0.26 for M2, 0.33 for M3, 0.35 for P2X1, 0.19 for P2X2, and 0.37 for P2X3. CONCLUSION: OnabotulinumtoxinA detrusor injections led to significant reductions in muscular M2, M3, P2X2, and P2X3 receptors. The reductions probably affect the generated force in the urinary bladder and could contribute to the clinically observed increase in residual urine.

Increase in cardiac M2-muscarinic receptor expression is regulated by GATA binding protein 4 (GATA-4) in streptozotocin-induced diabetic rats.

BACKGROUND: An increase in cardiac M2-muscarinic receptor (M2-mAChR) expression in diabetic rats has been observed, but the molecular mechanism of this increase remains unclear. The transcriptional activity of GATA binding protein 4 (GATA-4) has been documented to regulate the expression of M2-mAChR genes. In this study, we were interested in identifying the role of GATA-4 in the increase in M2-mAChR in diabetic rats and a primary culture of cardiomyocytes. METHODS: Streptozotocin-induced diabetic rats (STZ-rats) and high-glucose (D-glucose 30 mM, 24h)-treated primary cultures of cardiomyocytes from neonatal rats were used to investigate the role of GATA-4 in the change in M2-mAChR. The protein expression was determined by Western blot analysis. Phlorizin (Na(+)-glucose co-transport inhibitor), insulin, tiron (radical scavenger), PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor) were used. We also silenced GATA-4 by RNAi to investigate the changes in M2-mAChR expression. RESULTS: The cardiac output was reduced in STZ-rats with a higher expression of M2-mAChR or phosphorylated GATA-4 in the heart. These changes were reversed after correction of the blood sugar level. In cardiomyocytes, high glucose treatment also increased M2-mAChR expression and GATA-4 phosphorylation. These changes were reversed by tiron (ROS scavenger) or PD98059 (MEK/ERK inhibitor). However, an increase in M2-mAChR expression was not observed when GATA-4 was silenced by small interfering RNA (siRNA) in cardiomyocytes. CONCLUSIONS: We suggest that hyperglycemia can cause a higher expression of M2-mAChR in cardiomyocytes mainly through ROS to enhance MEK/ERK for phosphorylation of GATA-4.

Neuroprotection of green tea catechins on surgical menopause-induced overactive bladder in a rat model.

OBJECTIVE: A rat model of ovariectomy-induced voiding dysfunction has been established, which mimicked the urge incontinence in postmenopausal women. Previous studies have identified strong anti-inflammatory/antioxidant properties of green tea and its associated polyphenols. The aim of this study was to evaluate whether the green tea extract, epigallocatechin gallate (EGCG), could prevent an ovariectomy-induced overactive bladder. METHODS: The study included 48 female Sprague-Dawley rats, which were divided into four groups. After bilateral ovariectomy during the following 6-month period, 12 rats received an intraperitoneal injection of saline, 24 rats received either a low-dose (1 µM kg(-1) d(-1)) or a high-dose (10 µM kg(-1) d(-1)) EGCG intraperitoneal injection. The sham group consisted of twelve rats that were not ovariectomized. In vivo isovolumetric cystometrograms were performed in all groups before the animals were euthanized. The immunofluorescence study used neurofilament stains to evaluate intramural nerve damage. Western immunoblots and real-time polymerase chain reaction were performed to determine M2 and M3 muscarinic cholinergic receptors (MChRs) at both protein and messenger RNA (mRNA) expressions. RESULTS: Long-term ovariectomy significantly increased non-voiding contractions, whereas treatment with EGCG significantly attenuated the frequency of non-voiding contractions. Ovariectomy significantly decreased the numbers of neurofilament and increased M2 and M3 MChR protein and mRNA expressions. Treatment with EGCG restored the amount of neurofilament staining and decreased M2 and M3 MChR protein and mRNA overexpressions. CONCLUSIONS: This study confirmed that ovary hormone deficiency induced overactive bladder dysfunction via intramural nerve damage and muscarinic receptor overexpression. EGCG prevented ovariectomy-induced bladder dysfunction through neuroprotective effects in a dose-dependent fashion.

Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling.

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function.

Alterations of muscarinic acetylcholine receptors-2, 4 and α7-nicotinic acetylcholine receptor expression after ischaemia / reperfusion in the rat isolated heart.

1. Cardiac acetylcholine receptors are involved in the negative inotropic effect of the vagus and the protection of the stimulated vagal nerve against myocardial ischaemic injury. Acetylcholine receptors consist of five types of muscarinic acetylcholine receptors (M AChR) and several nicotinic acetylcholine receptors (nAChR). Notably, ischaemic heart disease is accompanied by substantial withdrawal of vagal activity. However, it is not entirely clear what the changes of M(2,4) AChR and α7-nAChR expression are after cardiac ischaemia/reperfusion (I/R) injury. 2. Cardiac functions were continuously recorded in Langendorff mode during 30 min of ischaemia and 60 min of reperfusion. Lactate dehydrogenase (LDH) leakage was measured. M(2,4) AChRs and α7-nAChR expression were measured by reverse transcription polymerase chain reaction and western blot. 3. In hearts exposed to I/R injury, left ventricular development pressure, heart rate and ± dP/dt decreased significantly compared with the controls. LDH leakage increased with respect to the controls during reperfusion. 4. In normal hearts, expression of M(2,4) AChR in the left ventricle were lower than in atria and the right ventricle, whereas expression of α7-nAChR was dramatically higher in the left ventricle and right ventricle than the atria. After reperfusion, the mRNA and protein expression of M(2) AChR increased notably in the left and right ventricle, and α7-nAChR was enhanced significantly in the left ventricle. M(4) AChR mRNA expression reduced notably after ischaemia and recovered to the control level after reperfusion in the atria, but the protein level did not change. 5. In conclusion, the increase in M(2) AChR and α7-nAChR after reperfusion might be the compensatory response to myocardial I/R injury, providing new information for treatment of myocardial I/R injury.

The impact of simulated birth trauma and ovariectomy on the gene expression of detrusor muscarinic receptors in female rats.

INTRODUCTION AND HYPOTHESIS: This study aims to investigate the effects of simulated birth trauma and ovariectomy on detrusor muscarinic receptors (M2 and M3), urethral neuronal nitric oxide synthase (nNOS), and estrogen receptor beta (ER beta). METHODS: Forty primiparous rats were equally divided into five groups: group A--delivery, group B--delivery plus ovariectomy, group C--delivery plus balloon dilatation for 2 h, group D--delivery plus balloon dilatation for 4 h, and group E--delivery plus balloon dilatation for 2 h plus ovariectomy. The gene expression of M2, M3, nNOS, and ER beta were assessed by reverse transcription polymerase chain reaction. RESULTS: Significant decreases in mRNA expression of M2 receptors and nNOS (P < 0.05), and a significant increase in M3 mRNA expression (P < 0.05) were observed in groups D and E when compared with group A. CONCLUSIONS: Ovariectomy following birth trauma may synergistically impact the function of urinary tract, this being possibly related to the modification of the gene expression of muscarinic receptors.

Expression of muscarinic M1 and M2 receptors in the anterior cingulate cortex associated with neuropathic pain.

The anterior cingulate cortex (ACC) and muscarinic receptors modulate pain. This study investigates changes in the expression of muscarinic-1 and -2 receptors (M1R, M2R) in rats' ACC (cg1-rostral- and cg2-caudal) using a model of neuropathic pain by denervation, measured as autotomy score (AS) for 8 days. Changes were analysed with painful stimuli and with scopolamine into the ACC prior to this scheme. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence to determine M1R and M2R's mRNA and protein levels, respectively. Animals were divided in low, medium and high AS groups. Cg1 showed decreased mRNA levels for both M1R and M2R in the low AS group, as opposed to an increased expression in the medium and high AS groups. Both receptors correlated positively with AS in these groups. In the scopolamine-treated animals there was an increase in mRNA levels for both receptors in cg1, whereas in cg2, mRNA levels of M1R decreased in all the AS and scopolamine groups. The increased M2R mRNA in cg2 correlated with AS in the low, medium and high AS groups whereas all the scopolamine groups showed an increase. Immunoreactivity of the M2R in cg1 decreased in the medium AS group in comparison to controls but scopolamine treatment produced an increase in the medium scopolamine AS group compared to the medium AS group. The M1R in cg1 and both receptors in cg2 showed no immunoreactivity changes. These results highlight the role of the M2R in cg1 related to the degree of autotomy.

Contrasting effects of allosteric and orthosteric agonists on m1 muscarinic acetylcholine receptor internalization and down-regulation.

A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M(1) mACh receptor internalization and down-regulation, as well as functional coupling in a Chinese hamster ovary (CHO) cell line. In contrast to full and partial orthosteric agonists, which cause significant receptor internalization and down-regulation, prolonged exposure to AC-42 did not significantly alter either cell-surface or total cellular M(1) mACh receptor expression. 77-LH-28-1, an AC-42 homolog, did cause some receptor internalization, but not down-regulation. The presence of atropine completely prevented the orthosteric agonist-induced adaptive changes in receptor populations; however, in contrast, the copresence of atropine and AC-42 significantly increased both cell-surface receptor and total M(1) mACh receptor expression. Maximal phosphoinositide hydrolysis responses to the partial agonist arecoline were similar in CHO-M(1) cells pretreated for 24 h with either AC-42 or vehicle; in contrast, these responses were markedly reduced when cells were pretreated with oxotremorine-M or pilocarpine. These data indicate that, whereas AC-42 binding to the M(1) mACh receptor can initiate signal transduction, the AC-42-liganded receptor is resistant to the usual mechanisms regulating receptor internalization and down-regulation. In addition, our data suggest unusual interactions between allosteric agonists and orthosteric antagonists to regulate cell-surface and total cellular receptor expression.

Differential novelty detection in rats selectively bred for novelty-seeking behavior.

"Novelty-seeking" behavior describes the variability of rats' locomotor response, namely high and low responders (HR and LR respectively), when exposed to a novel environment. Novelty-seeking in the rat is considered to model "sensation-seeking" in humans, a personality trait related to substance abuse. It is assumed that HR rats and LR rats differ in their emotional reactivity because of the disparate incentive value of contextual stimulus, thus differentially interacting with their environment. However, little is known about how HR and LR rats recognize novelty arising from the environment. The present study evaluates whether phenotype may affect spontaneous, non-spatial novelty discrimination. Selectively bred HR and LR rats were submitted to the novel-object recognition test. The task involved a delay of 3h after a first encounter with an object ("old"), which had to be discriminated from a second object ("new"). Object discrimination was assessed minute-by-minute during a 3-min choice session. Amnesic effects of scopolamine (0.5mg/kg, intraperitoneal) were also analyzed. HR-bred rats showed sustained novel-object recognition throughout the 3-min choice session, whereas LR-bred rats began to discriminate between objects only in the last minute. Surprisingly, level of discrimination in scopolamine-treated HR-bred rats was significant during the first minute of the choice test and diminished thereafter, presumably because both objects became equally familiar as they were explored. Additionally, scopolamine induced changes in muscarine M(2) receptor gene expression in a phenotype-dependent manner. Because consistent object discrimination mainly arises during the first minute, these findings may reflect differential novelty detection in HR-bred respect to LR-bred rats.

Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris.

N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand-receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1-10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B(max) value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.

M2 and M3-muscarinic acetylcholine receptors remodelling in patients with a dilated atrium.

BACKGROUND: Studies have shown that autonomic tone plays an important role in the pathogenesis of atrial fibrillation (AF) and remodelling of I(K, ACh) in chronic AF serves as a compensatory mechanism for the AF. The relation between atrial size and AF has been established. We investigated the remodelling of muscarinic receptors in patients with dilated atrium and assessed the relationship between the muscarinic receptor remodelling and the dilated atrium. METHODS: A small piece of the tip of the right atrial appendage was obtained from 19 patients in sinus rhythm (SR), 28 patients with mitral stenosis and 10 patients with chronic AF (AF > 6 months). Western-blot was used to determine the expression of M2 and M3 receptors. RESULTS: Patients with mitral stenosis and chronic AF had a larger left atrial diameter than patients in SR. The densities of the M2 receptor in patients with mitral stenosis (with AF and with SR) and chronic AF were lower than that in patients with SR (0.54 +/- 0.08 vs. 0.29 +/- 0.06, 0.26 +/- 0.05 and 0.28 +/- 0.06, P < 0.05). However, the densities of the M3 receptor in patients with mitral stenosis and chronic AF were higher than that in patients with SR (0.07 +/- 0.01 vs. 0.18 +/- 0.02, 0.17 +/- 0.01 and 0.15 +/- 0.01, P< 0.05). Furthermore, there was no significant difference in M2 and M3 receptors between AF and SR in patients with mitral stenosis. CONCLUSION: Remodelling of M2 and M3 receptors is not associated with AF, but with the dilated left atrium.