M1 Receptor Antagonist Research Articles

A comparison of scopolamine and biperiden as a rodent model for cholinergic cognitive impairment

RationaleThe nonselective muscarinic antagonist scopolamine hydrobromide (SCOP) is employed as the gold standard for inducing memory impairments in healthy humans and animals. However, its use remains controversial due to the wide spectrum of behavioral effects of this drug.ObjectiveThe present study investigated whether biperiden (BIP), a muscarinic m1 receptor antagonist, is to be preferred over SCOP as a pharmacological model for cholinergic memory deficits in rats. This was done by comparing the effects of SCOP and BIP using a battery of operant tasks: fixed ratio (FR5) and progressive ratio (PR10) schedules of reinforcement, an attention paradigm and delayed nonmatching to position task.ResultsSCOP induced diffuse behavioral disruption, which included sensorimotor responding (FR5, 0.3 and 1 mg/kg), food motivation (PR10, 1 mg/kg), attention (0.3 mg/kg, independent of stimulus duration), and short-term memory (delayed nonmatching to position (DNMTP), 0.1 and 0.3 mg/kg, delay-dependent but also impairment at the zero second delay). BIP induced relatively more selective deficits, as it slowed sensorimotor responding (FR5, 10 mg/kg) and disrupted short-term memory (DNMTP, 3 mg/kg, delay-dependent but no impairment at the zero second delay). BIP had no effect on food motivation (PR10) or attention.ConclusionMuscarinic m1 antagonists should be considered an interesting alternative for SCOP as a pharmacological model for cholinergic mnemonic deficits in animals.

BK Channels Regulate Contraction Secondary to M2 Muscarinic Acetylcholine Receptor Mediated Depolarization

Airway smooth muscles (ASM) contraction is primarily mediated by M2 and M3 muscarinic acetylcholine receptors. We and others have shown that one of the functions of large conductance Ca2+- and voltage-activated (BK) potassium channel and the accessory β1 subunit is to moderate ASM excitation-contraction coupling by opposing cholinergic evoked depolarization and reducing activation of L-type voltage-dependent calcium channels. Here, we have employed airway contraction studies, and a mouse knockout of the BK channel β1 subunit to investigate fuctional consequences specifically downstream of M2 signaling. Our results indicate that effects of BK channels are downstream of M2 signaling since M2 receptor antagonist (100 nM AF-DX 116) eliminates the enhanced contraction of β1 KO trachea. We found that the mechanism of M2 antagonist evoked relaxation of β1 KO is a reduction of excitation-contraction coupling since pretreatment with L-type Ca2+ channels blocker Nifedipine or with low extracellular potassium solution that hyperpolarizes membrane potential occludes effect of M2 antagonist. M2 mediated membrane depolarization and the role of BK/β1 in controlling ASM membrane potentials were directly confirmed using sharp electrode recording of membrane voltage. In summary, these results indicate that the important role of BK/β1 during M2 activation is to oppose membrane depolarization and thereby attenuating Ca2+-influx through voltage dependent Ca2+-channels. Blocking M2 signaling largely made BK channels superfluous to control of ASM contraction.

Involvement of decreased muscarinic receptor function in prepulse inhibition deficits in mice reared in social isolation

We have previously reported that galantamine, a weak acetylcholinesterase inhibitor, improves prepulse inhibition (PPI) deficits in mice reared in social isolation. ACh receptors are involved in the underlying mechanism of PPI, but whether rearing in social isolation causes dysfunction of the cholinergic system is unknown. In this study, we examined the involvement of muscarinic receptors in the improvement of PPI deficits induced by galantamine, and whether the cholinergic system is altered in mice reared in isolation. Three-week-old male ddY mice were housed in isolated cages for 6 weeks before the initiation of experiments to create PPI deficits. Cholinergic functions were determined by measuring the behavioural and neurochemical responses to nicotinic and muscarinic receptor agonists. The improvement by galantamine of social isolation-induced PPI deficits was blocked by scopolamine, a non-selective muscarinic antagonist, and telenzepine, a preferential M₁ receptor antagonist. Activation of M₁ receptors improved social isolation-induced PPI deficits. Social isolation did not affect choline acetyltransferase and acetylcholinesterase activities in the prefrontal cortex and hippocampus, but it reduced the locomotor-suppressive response to muscarinic agonist oxotremorine, but not to nicotine. The isolation also attenuated the M₁ receptor agonist N-desmethylclozapine-induced increase in prefrontal dopamine release. Galantamine improves PPI deficits of mice reared in social isolation via activation of M₁ receptors. Social isolation reduces the muscarinic, especially M₁, receptor function and this is involved in PPI deficits.

Changes in the Muscarinic Receptors on the Colonic Smooth Muscles of Rats with Spinal Cord Injury

ObjectiveTo investigate changes in (1) the colonic response to acetylcholine (Ach), (2) the muscarinic (M) receptors in the colon, and (3) the levels of colonic contraction-related proteins after a spinal cord injury (SCI).MethodWe divided 16 Sprague-Dawley rats into 2 groups: the control group and the SCI group. A spinal cord transection was performed surgically at the T10 vertebral level. After 1 week, the entire colon was divided into 2 segments, the proximal and distal colon. Each segment was mounted in a longitudinal or circular muscle direction in a 10-ml organ bath. We determined the intergroup differences as percentage changes in contractility after Ach treatment alone, Ach treatment with M2 receptor antagonist (AQ-RA741) pretreatment, and Ach treatment with M3 receptor antagonist (4-DAMP) pretreatment. Western blot analyses were performed to determine the expression level of RhoA, and heat shock protein 27 (HSP27).ResultsCompared to the control rats, the SCI rats showed an increased response to Ach along both the directions in the proximal colon (p<0.05). Compared to the control group, in the SCI group, the Ach response was significantly different in the proximal segment under AQ-RA741 pretreatment (p<0.05) and in the distal segment under 4-DAMP pretreatment (p<0.05). Findings of the western blot analyses showed a significant decrease in the level of protein gene product 9.5 in the proximal and distal colon and a significant increase in the level of RhoA and HSP27 in the proximal colon of the SCI rats.ConclusionOur results suggest that changes in colonic contractility after SCI are partly attributable to changes in the M receptor subtypes.

Opioid-Induced Constipation in Advanced Illness: Safety and Efficacy of Methylnaltrexone Bromide

Constipation, one of the major side effects of opiates used in palliative care, can impair patients’ quality of life to a point where it prevents sufficient pain control. Methylnaltrexone is a novel μ-receptor antagonist, which does not pass the blood brain barrier. It is licensed to treat opiate induced constipation for patients with advanced diseases. This review article presents an overview of pharmacology and safety of its application, evidence of its efficacy and economic aspects of its use in clinical practice. Available data are limited but strongly suggest that methylnaltrexone causes laxation in less than 24 hours for at least half of those patients over the first two weeks of usage without impairing pain control or causing serious adverse effects. To avoid danger of gastrointestinal perforation it is contraindicated for patients at risk for that complication. More research is needed to evaluate its long-term efficacy and economic impact.

The 5-HT2A Receptor Antagonist M100907 Produces Antiparkinsonian Effects and Decreases Striatal Glutamate

5-HT plays a regulatory role in voluntary movements of the basal ganglia and has a major impact on disorders of the basal ganglia such as Parkinson's disease (PD). Clinical studies have suggested that 5-HT2 receptor antagonists may be useful in the treatment of the motor symptoms of PD. We hypothesized that 5-HT2A receptor antagonists may restore motor function by regulating glutamatergic activity in the striatum. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exhibited decreased performance on the beam-walking apparatus. Peripheral administration of the 5-HT2A receptor antagonist M100907 improved performance of MPTP-treated mice on the beam-walking apparatus. In vivo microdialysis revealed an increase in striatal extracellular glutamate in MPTP-treated mice and local perfusion of M100907 into the dorsal striatum significantly decreased extracellular glutamate levels in saline and MPTP-treated mice. Our studies suggest that blockade of 5-HT2A receptors may represent a novel therapeutic target for the motor symptoms of PD.

Muscarinic receptor antagonist therapy improves acute pulmonary dysfunction after smoke inhalation injury in sheep

Inhalation injury contributes to the morbidity and mortality of burn victims. In humans and in an ovine model of combined smoke inhalation and burn injury, bronchospasm and acute airway obstruction contribute to progressive pulmonary insufficiency. This study tests the hypothesis that muscarinic receptor antagonist therapy with tiotropium bromide, an M1 and M3 muscarinic receptor antagonist, will decrease the airway constrictive response and acute bronchial obstruction to improve pulmonary function compared to injured animals without treatment. Randomized, prospective study involving 32 sheep. Large-animal intensive care research laboratory. The study consisted of six groups: a sham group (n=4, instrumented noninjured), a control group (n=6, injured and not treated), and tiotropium bromide-treated groups, including both preinjury and postinjury nebulization protocols. Treatments for these groups included nebulization with 36 μg of tiotropium bromide 1 hr before injury (n=6) and postinjury nebulization protocols of 18 μg (n=6), 36 μg (n=6), and 72 μg (n=4) administered 1 hr after injury. All treated groups received an additional 14.4 μg every 4 hrs for the 24-hr study period. Pretreatment with tiotropium bromide significantly attenuated the increases in ventilatory pressures, pulmonary dysfunction, and upper airway obstruction that occur after combined smoke inhalation and burn injury. Postinjury treatments with tiotropium bromide were as effective as pretreatment in preventing pulmonary insufficiency, although a trend toward decreased obstruction was present only in all post-treatment conditions. There was no improvement noted in pulmonary function in animals that received a higher dose of tiotropium bromide. This study describes a contribution of acetylcholine to the airway constrictive and lumenal obstructive response after inhalation injury and identifies low-dose nebulization of tiotropium bromide as a potentially efficacious therapy for burn patients with severe inhalation injury.

Creating the animated intensive care unit

Critical care medicine has matured greatly as a field in the past decade. Much has been learned concerning the institution of life support therapies to sustain patients with diverse and multiple organ failures, thus providing patients with a window of opportunity to recover from potentially life-ending insults. The management of critically ill patients has increasingly involved creation of a highly controlled environment by care providers, with patients immobilized, tethered to devices, and receiving multiple drugs to facilitate the entire process. Although it has been assumed that such control of the patient has been necessary to implement essential therapies and to tailor life support systems such as mechanical ventilation, this assumption may be unfounded or at least overplayed, as knowledge of the adverse effects of this approach have been identified and quantified. Extant information, based on observational studies and a few interventional trials, would suggest a radically different approach to care is warranted, even given the difficulties in reversing the current culture of critical care management. Specifically, methods to avoid entirely, or minimize, neuromuscular blockade and sedation are supported by recent literature. These methods include the use of noninvasive ventilation in appropriately selected patients, the development of mechanical ventilators more synchronous with patient efforts and needs, and the use of sedation strategies to avoid drug accumulations with protracted effects. These methods, in turn, afford opportunities to avoid extreme immobilization and institute physiotherapy earlier than previously had been thought possible. In addition to the neuropsychiatric and neuromuscular benefits that could derive from minimizing opiate administration in critically ill patients, gut hypomotility could be avoided. This, in turn, could facilitate earlier and more complete enteral nutrition. Even when opioids have to be administered in generous amounts for control of pain that may accompany critical illness, it is now possible to block the peripheral actions of these medications with the μ-receptor antagonist methylnaltrexone. Other new drugs being introduced into the critical care unit such as dexmedetomidine may also provide a greater ability to achieve analgesia and anxiolysis without some of the adverse concomitant effects seen with more traditional drug regimens. The ultimate goal of this multipronged program to facilitate the maintenance of patients who are more interactive with their care providers, and the life support provided in the intensive care unit would be to speed the pace of recovery and to diminish the need for the protracted rehabilitation that often follows survival from critical illness.

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

Flp-In(TM) T-REx(TM) 293 cells expressing a wild type human M(3) muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M(3) receptor expressed stably in Flp-In(TM) TREx(TM) 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC(50) values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M(3) muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.

The α 2 adrenergic receptor antagonist idazoxan, but not the serotonin-2A receptor antagonist M100907, partially attenuated reward deficits associated with nicotine, but not amphetamine, withdrawal in rats

Based on phenomenological similarities between anhedonia (reward deficits) associated with drug withdrawal and the negative symptoms of schizophrenia, we showed previously that the atypical antipsychotic clozapine attenuated reward deficits associated with psychostimulant withdrawal. Antagonism of α 2 adrenergic and 5-HT 2A receptors may contribute to these effects of clozapine. We investigated here whether blockade of α 2 or 5-HT 2A receptors by idazoxan and M100907, respectively, would reverse anhedonic aspects of psychostimulant withdrawal. Idazoxan treatment facilitated recovery from spontaneous nicotine, but not amphetamine, withdrawal by attenuating reward deficits and increase the number of somatic signs. Thus, α 2 adrenoceptor blockade may have beneficial effects against nicotine withdrawal and may be involved in the effects of clozapine previously observed. M100907 worsened the anhedonia associated with nicotine and amphetamine withdrawal, suggesting that monotherapy with M100907 may exacerbate the expression of the negative symptoms of schizophrenia or nicotine withdrawal symptoms in people, including schizophrenia patients, attempting to quit smoking.

Neuronal localization of m1 muscarinic receptor immunoreactivity in the rat basolateral amygdala.

Muscarinic cholinergic neurotransmission in the basolateral nuclear complex (BLC) of the amygdala is critical for memory consolidation in emotional/motivational learning tasks. Although knowledge of the localization of muscarinic receptor subtypes in the BLC would contribute to an understanding of the actions of acetylcholine in mnemonic function, previous receptor binding and in situ hybridization studies lacked the resolution necessary to identify which neurons in the BLC express different receptor subtypes. In the present study immunohistochemistry was used to study the neuronal localization of the m1 receptor. The intensity of m1 immunoreactivity varied in different nuclei of the amygdala, and was most robust in the BLC, and in the adjacent posterolateral cortical nucleus. The density and morphology of labeled neurons in the BLC suggested that the m1+ neuronal population included pyramidal cells, the principal neurons in this amygdalar region. In addition, there was dense punctate m1 immunoreactivity in the neuropil of the BLC. Dual labeling immunofluorescence studies of the BLC using antibodies to cell type specific markers were performed to more definitively determine the phenotype of m1-positive (m1+) neurons. An antibody to calcium/calmodulin protein kinase II (CaMK) was used to label pyramidal cells, whereas an antibody to glutamic acid decarboxylase was used to label interneurons. Virtually all of the intensely labeled m1+ neurons of the BLC were CaMK+ pyramidal cells. These data suggest that the ability of M1 receptor antagonists to impair memory consolidation in the BLC is mainly due to blockade of cholinergic influences on the activity of pyramidal neurons.

Retrograde facilitation of verbal memory by trihexyphenidyl in healthy elderly with and without the APOE ε4 allele

Retrograde facilitation (RF) of information learned prior to acute oral administration of trihexyphenidyl, a preferential muscarinic M1 receptor antagonist which impairs new learning, was studied in 24 healthy elderly subjects. The relationship between the RF induced by this anticholinergic drug and the APOE epsilon4 allele was also examined. Acute adverse performance effects of trihexyphenidyl (1- and 2mg) were determined using the Buschke Selective Reminding Test administered pre-drug and at 1, 2.5, and 5h post-drug. Recall of pre-drug words at the end of the fifth hour neuropsychological assessment (end-of-session recall) was of primary interest. Words studied before drug administration were better recalled following 2mg trihexyphenidyl compared to placebo, and this RF effect was not affected by the APOE epsilon4 allele. Better recall of pre-drug words following 2-mg trihexyphenidyl was associated with a greater amnestic effect of this dose. Our findings demonstrated that RF induced by trihexyphenidyl was related to anterograde amnestic effects of the drug and resulted in part from drug-induced reduction of retroactive interference.

5-HT2A receptor antagonists improve motor impairments in the MPTP mouse model of Parkinson’s disease

Clinical observations have suggested that ritanserin, a 5-HT(2A/C) receptor antagonist may reduce motor deficits in persons with Parkinson's Disease (PD). To better understand the potential antiparkinsonian actions of ritanserin, we compared the effects of ritanserin with the selective 5-HT(2A) receptor antagonist M100907 and the selective 5-HT(2C) receptor antagonist SB 206553 on motor impairments in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-treated mice exhibited decreased performance on the beam-walking apparatus. These motor deficits were reversed by acute treatment with L-3,4-dihydroxyphenylalanine (levodopa). Both the mixed 5-HT(2A/C) antagonist ritanserin and the selective 5-HT(2A) antagonist M100907 improved motor performance on the beam-walking apparatus. In contrast, SB 206553 was ineffective in improving the motor deficits in MPTP-treated mice. These data suggest that 5-HT(2A) receptor antagonists may represent a novel approach to ameliorate motor symptoms of Parkinson's disease.

Differential effects of serotonin (5‐HT)2 receptor‐targeting ligands on locomotor responses to nicotine‐repeated treatment

We verified the hypothesis that serotonin (5-HT)(2) receptors control the locomotor effects of nicotine (0.4 mg kg(-1)) in rats by using the 5-HT(2A) receptor antagonist M100907, the preferential 5-HT(2A) receptor agonist DOI, the 5-HT(2C) receptor antagonist SB 242084, and the 5-HT(2C) receptor agonists Ro 60-0175 and WAY 163909. Repeated pairings of a test environment with nicotine for 5 days, on Day 10 significantly augmented the locomotor activity following nicotine administration. Of the investigated 5-HT(2) receptor ligands, M100907 (2 mg kg(-1)) or DOI (1 mg kg(-1)) administered during the first 5 days in combination with nicotine attenuated or enhanced, respectively, the development of nicotine sensitization. Given acutely on Day 10, M100907 (2 mg kg(-1)), Ro 60-0175 (1 mg kg(-1)), and WAY 163909 (1.5 mg kg(-1)) decreased the expression of nicotine sensitization. In another set of experiments, where the nicotine challenge test was performed on Day 15 in animals treated repeatedly (Days: 1-5, 10) with nicotine, none of 5-HT(2) receptor ligands administered during the second withdrawal period (Days: 11-14) to nicotine-treated rats altered the sensitizing effect of nicotine given on Day 15. Our data indicate that 5-HT(2A) receptors (but not 5-HT(2C) receptors) play a permissive role in the sensitizing effects of nicotine, while stimulation of 5-HT(2A) receptors enhances the development of nicotine sensitization and activation of 5-HT(2C) receptors is essential for the expression of nicotine sensitization. Repeated treatment with the 5-HT(2) receptor ligands within the second nicotine withdrawal does not inhibit previously established sensitization.

Muscarinic acetylcholine receptors operative in mouse taste cells

A heterogeneous population of taste bud cells includes cells of three different types (type I to type III) that serve distinctive physiological functions. Evidence implicates several signaling systems in mediating cell-to-cell communications in the taste bud. In particular, a variety of receptors and their ligands known as classical neurotransmitters/neuromodulators have been identified in the taste tissue. Although potentially, they all can be involved in extracellular signaling, their significance for taste bud physiology is not well understood. Here we focused on the cholinergic system to unveil whether one operates in the mouse taste bud, and if so, to elucidate taste cells functionally expressing ACh receptors. Taste cells identified electrophysiologically were stimulated by the non-hydrolizable ACh analog carbachol and their responsiveness was evaluated by Ca2+ transients elicited by this agonist. Carbachol stimulated cellular responses in a wide range of concentrations, exhibiting EC50 = 2.5 μM on average. Carbachol-elicited Ca2+ transients were observed in the taste cell cytoplasm even with 100 nM Ca2+ in the bath, while 1 μM atropine, a muscarinic receptor antagonist, completely suppressed cellular responses. Together, these observations and negligible responses to nicotine pointed out that muscarinic receptors predominantly mediated the carabachol-dependent Ca2+ mobilization in taste cells. Notably, telenzipine, a subtype-specific antagonist of M1-receptors, inhibited carbachol responses only partially. Consistently, the M1-agonist McN-A-343 was much less effective than carbachol. These findings indicate that although M1-receptors are functional in mouse taste cells, muscarinic receptors of other types, most likely M3 and/or M5, are largely responsible for Ca2+ transients elicited by carbachol. The analysis of expression of muscarinic receptor genes with RT-PCR revealed transcripts for M1-, M3-, and M5-isoforms in preparations of the taste tissue. It thus appears that solely type I cells are responsive to cholinergic agonists and recognize those by employing multiple muscarinic receptors coupled to the phosphoinositide cascade.

New insights into molecular targets for urinary incontinence.

Urinary incontinence (UI) is a disease affecting quality of life of 200 million patients worldwide. It is characterized by involuntary loss of urine. The factors involved are cystitis, detrusor hyperreflexia, spinal injury, benign prostatic hyperplasia, etc. The surge in the number of reviews on this subject indicates the amount of research devoted to this field. The prevalence is increasing at an alarming rate but unfortunately, only a few medications are currently available for this condition. There are peripheral as well as central targets including cholinergic, vanilloid, prostaglandin, kinin, calcium channel, cannabinoid, serotonin, and GABA-receptors, which act by different mechanisms to treat different types of incontinence. Drugs acting on the central nervous system (CNS) increase urinary bladder capacity, volume, or pressure threshold for micturition reflex activation while peripherally acting drugs decrease the amplitude of micturition contraction and residual volume. Anticholinergic drugs specifically M3 receptor antagonists are the first choice but have frequent side effects such as dry mouth, CNS disturbances, etc. Therefore, there is a need to understand the biochemical pathways that control urinary dysfunction to determine the potential to which they can be exploited in the treatment of this condition. This article reviews the central and peripheral molecular targets and the potential therapeutic approaches to the treatment of UI.

Functional expression of muscarinic and purinoceptors in the urinary bladder of male and female rats and guinea pigs

The objectives of this study were to compare the functional expression of muscarinic and purinergic receptors in the urinary bladder of 2 species, rat and guinea pig under comparable experimental conditions; and to test whether the receptors in males and females differ. Reverse transcription-polymerase chain reaction (RT-PCR) techniques were used to identify gene expression profiles in bladder smooth muscle (total n=8 rats, 7 guinea pigs) and mechanical responses to nerve stimulation and applied acetylcholine (ACh) in the presence of specific antagonists were used to identify functional receptor sub-types (total n=12 rats, 16 guinea pigs). RT-PCR indicated that M2 and M3 were the predominant muscarinic receptor genes in both the male and female rat and guinea pig bladders. The phasic component of the nerve-induced contraction was greater in guinea pigs vs. rats. The tonic component and the ACh response were inhibited by the M3 receptor antagonist, darifenacin (10(-6) M, P≤0.05), but not by the M2 receptor antagonist, methoctramine (10(-5) M). The antipurinergic drug α, β-methylene ATP (5 × (-5) M) caused a significant reduction in the amplitude of the phasic response to nerve stimulation in all groups, and this effect was significantly greater in male vs. female rats. mRNA for the purinergic P2X1, P2X2, P2X4, P2X5 and P2X7 receptors was detected in both male and female rats, whereas P2X3 and P2X6 were inconsistently detected in male rats. The P2X1 purinoceptor antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4', 8'-disulphonate) (PPNDS), only inhibited nerve induced contractions at high concentrations (up to 10(-4) M). While only minor functional differences were documented in cholinergic and purinergic bladder contractile responses between male and female animals, and between rats and guinea pigs, data such as presented in this study are critical in determining how relative functional contributions may change in the diseased state, providing valuable information towards new treatment options.

Subarachnoid transplantation of immortalized galanin-overexpressing astrocytes attenuates chronic neuropathic pain

Treatment of chronic neuropathic pain resulted from peripheral nerve injury is one of the most difficult problems in modern clinical practice. The use of cell lines as biologic “minipumps” to chronically deliver anti-nociceptive molecules into the pain-processing centers of spinal cord is a newly developing technique for the treatment of pain. Moreover, spinal administration of exogenous galanin (GAL) is a useful target for the treatment of chronic pain after nerve injury. Because of better histocompatibility, lower immunogenicity and reproducibility, immortalized astrocytes (IAST) have been served as a promising cellular vehicle to deliver therapeutic molecules into CNS. In this study, the rat IAST was transfected with rat preprogalanin cDNA and the galanin-synthesizing and secreting cell line, IAST/GAL, was isolated. After cells were transplanted into the subarachnoid space of rats with chronic neuropathic pain induced by spared nerve injury (SNI) of sciatic nerve, their analgesic potential was evaluated by behavioral tests. The results showed that IAST/GAL transfected with preprogalanin gene could express and secrete significantly higher level of GAL protein in vitro and in vivo as compared with control cells. In addition, the pain-related behaviors, thermal hyperalgesia and mechanical allodynia were significantly alleviated during the 1–7 weeks after grafts of IAST/GAL cells, which could be reversed by galanin receptor antagonist M35 temporarily. Taken together, these data suggest that subarachnoid transplant of immortalized galanin-overexpressing astrocytes near the pain-processing centers was able to reverse the development of chronic neuropathic pain, which offers an adjunct approach to currently used therapies for the pain management.

Cholinergic Modulation Differs between Basal and Apical Dendritic Excitation of Hippocampal CA1 Pyramidal Cells

We hypothesize that endogenous cholinergic modulation of dendritic processing of hippocampal CA1 is layer specific, and it specifically enhances spike output resulting from basal as compared with the apical dendritic excitation. Laminar profiles of evoked field potentials were recorded in the CA1 area of urethane-anesthetized rats using multichannel silicon probes and analyzed as current source density. High-frequency stimulation of the pontis oralis (PnO) attenuated the midapical more than the basal or distal apical dendritic excitatory sink. Population spike (PS) and excitatory sink-PS potentiation resulting from basal dendritic excitation were facilitated, while the PS evoked by apical dendritic stimulation was attenuated by PnO stimulation. Perfusion of cholinergic agonist carbachol onto hippocampal slices in vitro also attenuated the apical more than the basal dendritic excitatory postsynaptic potentials. Excitatory sink attenuation and PS changes after PnO stimulation were blocked by systemic or local scopolamine and by intracerebroventricular (icv) M1 receptor antagonist pirenzepine but not by icv M2 receptor antagonist AFDX-116 or nicotinic antagonists. However, a hippocampal theta rhythm activated by PnO stimulation was blocked by systemic but not by local scopolamine. We conclude that endogenous acetylcholine mediates a stronger presynaptic inhibition of the midapical than basal and distal apical excitation mainly through M1 receptors.

Pain relief by spinal cord stimulation involves serotonergic mechanisms: An experimental study in a rat model of mononeuropathy

The aim of the present study was to examine the role of the spinal serotonergic system in the pain relieving effect of spinal cord stimulation (SCS) using a rat model of mononeuropathy. Tactile withdrawal thresholds, cold responses and heat withdrawal latencies were assessed before and after SCS. In some rats, SCS produced an attenuation of the hypersensitivity following nerve injury (SCS responding rats). When SCS was applied immediately prior to sacrifice, the 5-HT content in the dorsal quadrant of the spinal cord ipsilateral to the nerve injury was increased in SCS responding rats. But there was no change in responding rats without stimulation, or in SCS non-responding rats with or without stimulation or in controls. Immunohistochemical examination showed a high density of 5-HT stained terminals in the dorsal horn superficial laminae (I-II) in SCS responding rats following stimulation. It was also found that i.t. administration of a sub-effective dose of serotonin in SCS non-responding rats markedly enhanced the pain relieving effect of SCS on tactile and cold hypersensitivity, while there was no effect on heat hyperalgesia. This enhanced effect on tactile hypersensitivity could be partially blocked by a GABAB receptor antagonist (CGP 35348) but not by a muscarinic M4 receptor antagonist (Muscarinic toxin 3) administered i.t. shortly before the 5-HT injection. In conclusion, there is evidence that the spinal 5-HT system plays an important role in the mode of action of SCS involving the activation of descending serotonergic pathways that may inhibit spinal nociceptive processing partially via a GABAergic link.