The interaction between hippocampal cholinergic and nitrergic neurotransmission coordinates NMDA-dependent behavior and autonomic changes induced by contextual fear retrieval.

RATIONALE: Re-exposing an animal to an environment previously paired with an aversive stimulus evokes large alterations in behavioral and cardiovascular parameters. Dorsal hippocampus (dHC) receives important cholinergic inputs from the basal forebrain, and respective acetylcholine (ACh) levels are described to influence defensive behavior. Activation of muscarinic M1 and M3 receptors facilitates autonomic and behavioral responses along threats. Evidence show activation of cholinergic receptors promoting formation of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) in dHC. Altogether, the action of ACh and NO on conditioned responses appears to converge within dHC. OBJECTIVES: As answer about how ACh and NO interact to modulate defensive responses has so far been barely addressed, we aimed to shed additional light on this topic. METHODS: Male Wistar rats had guide cannula implanted into the dHC before being submitted to the contextual fear conditioning (3footshocks/085 mA/2 s). A catheter was implanted in the femoral artery the next day for cardiovascular recordings. Drugs were delivered into dHC 10 min before contextual re-exposure, which occurred 48 h after the conditioning procedure. RESULTS: Neostigmine (Neo) amplified the retrieval of conditioned responses. Neo effects (1 nmol) were prevented by the prior infusion of a M1-M3 antagonist (fumarate), a neuronal nitric oxide synthase inhibitor (NPLA), a NO scavenger (cPTIO), a guanylyl cyclase inhibitor (ODQ), and a NMDA antagonist (AP-7). Pretreatment with a selective M1 antagonist (pirenzepine) only prevented the increase in autonomic responses induced by Neo. CONCLUSION: The results show that modulation in the retrieval of contextual fear responses involves coordination of the dHC M1-M3/NO/cGMP/NMDA pathway.

Antagonism of the Muscarinic Acetylcholine Type 1 Receptor Enhances Mitochondrial Membrane Potential and Expression of Respiratory Chain Components via AMPK in Human Neuroblastoma SH-SY5Y Cells and Primary Neurons.

Impairments in mitochondrial physiology play a role in the progression of multiple neurodegenerative conditions, including peripheral neuropathy in diabetes. Blockade of muscarinic acetylcholine type 1 receptor (M1R) with specific/selective antagonists prevented mitochondrial dysfunction and reversed nerve degeneration in in vitro and in vivo models of peripheral neuropathy. Specifically, in type 1 and type 2 models of diabetes, inhibition of M1R using pirenzepine or muscarinic toxin 7 (MT7) induced AMP-activated protein kinase (AMPK) activity in dorsal root ganglia (DRG) and prevented sensory abnormalities and distal nerve fiber loss. The human neuroblastoma SH-SY5Y cell line has been extensively used as an in vitro model system to study mechanisms of neurodegeneration in DRG neurons and other neuronal sub-types. Here, we tested the hypothesis that pirenzepine or MT7 enhance AMPK activity and via this pathway augment mitochondrial function in SH-SY5Y cells. M1R expression was confirmed by utilizing a fluorescent dye, ATTO590-labeled MT7, that exhibits great specificity for this receptor. M1R antagonist treatment in SH-SY5Y culture increased AMPK phosphorylation and mitochondrial protein expression (OXPHOS). Mitochondrial membrane potential (MMP) was augmented in pirenzepine and MT7 treated cultured SH-SY5Y cells and DRG neurons. Compound C or AMPK-specific siRNA suppressed pirenzepine or MT7-induced elevation of OXPHOS expression and MMP. Moreover, muscarinic antagonists induced hyperpolarization by activating the M-current and, thus, suppressed neuronal excitability. These results reveal that negative regulation of this M1R-dependent pathway could represent a potential therapeutic target to elevate AMPK activity, enhance mitochondrial function, suppress neuropathic pain, and enhance nerve repair in peripheral neuropathy.

Unexpected scaffold rearrangement product of pirenzepine found in commercial samples.

Pharmacovigilance aims at a better understanding of the molecular events triggered by medications to prevent adverse effects, which despite significant advances in our analytical repertoire plague the use of drugs until today. In this study, we find that clinically prescribed and commercially available pirenzepine may not be the correct compound. Pirenzepine can undergo an unexpected scaffold rearrangement from the pharmaceutical active ingredient (API) to a previously uncharacterized benzimidazole. The rearrangement occurs under highly acidic conditions, which were believed to favour the dihydrochloride formation of pirenzepine. The rearranged products of pirenzepine and the structurally related telenzepine have significantly decreased affinity for the muscarinic acetylcholine receptor, the pharmacological target of these compounds. Fortunately, in situ rearrangement after oral application is no safety issue, as we show that reaction kinetics in gastric acid prevent rearrangement. The research community should consider appropriate measures to perform reliable receiving inspections in the commercial supply of well described and frequently used chemicals, in particular if experiments yield unexpected results.

Transcriptome-based insights into gene networks controlling myopia prevention.

Myopia (short-sightedness), usually caused by excessive elongation of the eye during development, has reached epidemic proportions worldwide. In animal systems including the chicken model, several treatments have been shown to inhibit ocular elongation and experimental myopia. Although diverse in their apparent mechanism of action, each one leads to a reduction in the rate of ocular growth. We hypothesize that a defined set of retinal molecular changes may underlie growth inhibition, irrespective of the treatment agent used. Accordingly, across five well-established but diverse methods of inhibiting myopia, significant overlap is seen in the retinal transcriptome profile (transcript levels and alternative splicing events) in chicks when analyzed by RNA-seq. Within the two major pathway networks enriched during growth inhibition, that of cell signaling and circadian entrainment, transcription factors form the largest functional grouping. Importantly, a large percentage of those genes forming the defined retinal response are downstream targets of the transcription factor EGR1 which itself shows a universal response to all five growth-inhibitory treatments. This supports EGR1's previously implicated role in ocular growth regulation. Finally, by contrasting our data with human linkage and GWAS studies on refractive error, we confirm the applicability of our study to the human condition. Together, these findings suggest that a universal set of transcriptome changes, which sit within a well-defined retinal network that cannot be bypassed, is fundamental to growth regulation, thus paving a way for designing novel targets for myopia therapies.

Monitoring Intracellular Calcium in Response to GPCR Activation: Comparison Between Microtiter Plates and Microfluidic Assays.

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).

Rational Design of Photochromic Analogues of Tricyclic Drugs.

Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo. Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine-2, maintained a moderate but promising M1 vs M2 subtype selectivity. These photoswitchable "crypto-azologs" of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.

Effects of pirenzepine on vonoprazan-induced gastric acid inhibition and hypergastrinemia.

BACKGROUND: Compared to proton pump inhibitors, vonoprazan exerts a greater inhibitory effect on gastric acid secretion and is useful for treating acid-related diseases, such as gastro-esophageal reflux disease. However, there is a problem that vonoprazan causes hypergastrinemia, which confers a risk of carcinoid tumor. A previous report demonstrated that pirenzepine, an M1 muscarinic receptor antagonist, enhances the acid inhibitory effects while suppressing hypergastrinemia induced by omeprazole. Here, we examined whether pirenzepine enhances the gastric acid inhibitory effects of vonoprazan without further increasing serum gastrin levels. METHODS: Eleven healthy volunteers were subjected to 24-h intragastric pH monitoring and serum gastrin measurements on day 7 of three different regimens: pirenzepine 75 mg alone, vonoprazan 10 mg alone, and vonoprazan 10 mg plus pirenzepine 75 mg administered in a randomized crossover fashion. RESULTS: Median pH 4 holding time ratios (range) achieved with pirenzepine 75 mg, vonoprazan 10 mg, and vonoprazan 10 mg plus pirenzepine 75 mg were 6.9% (2.4-32.8%), 88.4% (54.6-100%), and 84.2% (40.3-100%), respectively. Respective serum gastrin levels were 79 (75-210) pg/ml, 310 (110-870) pg/ml, and 170 (140-930) pg/ml. In cases with hypergastrinemia (gastrin ≥ 200 pg/ml) induced by vonoprazan 10 mg alone, concomitant treatment with pirenzepine significantly reduced serum gastrin levels from 370 to 180 pg/ml (P = 0.028). CONCLUSION: Although pirenzepine does not enhance acid inhibition, it does improve hypergastrinemia induced by vonoprazan to some extent.

Muscarinic M1, but not M4, receptor antagonism impairs divided attention in male rats.

Divided attention may be more important than ever to comprehend, given ubiquitous distractors in modern living. In humans, concern has been expressed about the negative impact of distraction in education, the home, and the workplace. While acetylcholine supports divided attention, in part via muscarinic receptors, little is known about the specific muscarinic subtypes that may contribute. We designed a novel, high-response rate test of auditory sustained attention, in which rats complete variable-ratio runs on one of two levers, rather than emitting a single response. By doing this, we can present a secondary visual distractor task during some trials, for which a correct nosepoke response is reinforced with a more palatable food pellet. The nonspecific muscarinic antagonist scopolamine impaired performance, and slowed and reduced lever press activity. We then explored antagonists that preferentially block the M1 and M4 subtypes, because these receptors are potential therapeutic targets for cognitive enhancers. Telenzepine, an M1-preferring antagonist, impaired divided attention performance, but not performance of the attention task without distraction. Telenzepine also had fewer nonspecific effects than scopolamine. In contrast, the M4-preferring antagonist tropicamide had no effects. Analysis of overall behavior also indicated that accuracy in the main attention task decreased as a function of engagement with the distractor task. These results implicate the M1 receptor in divided attention.

Critical role of hippocampal muscarinic acetylcholine receptors on memory reconsolidation in mice.

Over the years, experimental and clinical evidence has given support to the idea that acetylcholine (Ach) plays an essential role in mnemonic phenomena. On the other hand, the Hippocampus is already known to have a key role in learning and memory. What is yet unclear is how the Ach receptors may contribute to this brain region role during memory retrieval. The Ach receptors are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors and the metabotropic muscarinic acetylcholine receptors. Back in 2010, we demonstrated for the first time the critical role of hippocampal α7 nicotinic acetylcholine receptors in memory reconsolidation process of an inhibitory avoidance response in mice. In the present work, we further investigate the possible implication of hippocampal muscarinic Ach receptors (mAchRs) in this process using a pharmacological approach. By specifically administrating agonists and antagonists of the different mAchRs subtypes in the hippocampus, we found that M1 and M2 but not M3 subtype may be involved in memory reconsolidation processes in mice.

Activation of cortical M1 muscarinic receptors and related intracellular signaling is necessary for reactivation-induced object memory updating.

Reactivated long-term memories can become labile and sensitive to modification. Memories in this destabilized state can be weakened or strengthened, but there is limited research characterizing the mechanisms underlying retrieval-induced qualitative updates (i.e., information integration). We have previously implicated cholinergic transmission in object memory destabilization. Here we present a novel rodent paradigm developed to assess the role of this cholinergic mechanism in qualitative object memory updating. The post-reactivation object memory modification (PROMM) task exposes rats to contextual information following object memory reactivation. Subsequent object exploratory performance suggests that the contextual information is integrated with the original memory in a reactivation- and time-dependent manner. This effect is blocked by interference with M1 muscarinic receptors and several downstream signals in perirhinal cortex. These findings therefore demonstrate a hitherto unacknowledged cognitive function for acetylcholine with important implications for understanding the dynamic nature of long-term memory storage in the normal and aging brain.

Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase ß to Augment Mitochondrial Function and Prevent Neurodegeneration.

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M1R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKß to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M1R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 µM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 µM) or short hairpin RNA to CaMKKß. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 µM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M1R-modulated, CaMKKß-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

Participation of acetylcholine and its receptors in the contractility of inflamed porcine uterus.

This study analyzed the effect of inflammation on acetylcholine (ACh)-induced muscarinic receptors (MR)2 and MR3 conducted contractility of the porcine uterus. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) was injected into uterine horns or laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. Compared to the period before ACh treatment, ACh (10-5 M) increased the tension in myometrium (MYO) and endometrium/myometrium (ENDO/MYO) of the CON group (P < 0.01) and in ENDO/MYO of the SAL group (P < 0.01), the amplitude in strips of the CON (P < 0.05) and SAL (MYO: P < 0.05, ENDO/MYO: P < 0.001) groups and the frequency in strips of the CON (MYO: P < 0.01, ENDO/MYO: P < 0.001) and SAL (P < 0.01) groups. In the E.coli group, ACh (10-5 M) reduced the amplitude in MYO (P < 0.05) and ENDO/MYO (P < 0.001), increased the frequency in MYO (P < 0.01) and ENDO/MYO (P < 0.001) and did not change (P > 0.05) the tension. ACh (10-5 M) in ENDO/MYO of the E.coli group, reduced the tension compared to the CON group (P < 0.05) and the amplitude compared to other groups (P < 0.001), while increased the frequency in relation to the SAL group (P < 0.05). MR2 antagonist (AF-DX 44 116) and ACh (10-5 M) reduced (by 16.92%, P < 0.01) the tension in MYO of the CON group and increased (P < 0.01) it in the E.coli group compared to the period before antagonist and ACh addition. In MYO of the SAL group, the tension was increased (P < 0.01) in response to MR3 antagonist (4-DAMP) and ACh (10-7, 10-6 M). In the E.coli group, these substances did not change (P > 0.05) the tension, but it was lower (P < 0.001) in MYO (ACh: 10-7 M) and ENDO/MYO (ACh: 10-5 M) than in the SAL group. MR2 or MR3 antagonists and ACh (10-5 M) increased (P < 0.05-0.001) the amplitude in strips of the CON and SAL groups and reduced it in the E.coli group (P < 0.001) compared to the period before antagonists and ACh use. This parameter in the E.coli group was lower (P < 0.001) after using MR2 or MR3 antagonists and ACh (10-6, 10-5 M) than in other groups. Both antagonists and ACh (10-5 M) reduced the frequency in the CON, SAL (P < 0.05) and E.coli (MR2 antagonist: P < 0.01, MR3 antagonist: P < 0.05) groups compared to period before antagonists and ACh addition. Data show that ACh reduces the contractility of the inflamed porcine uterus by MR2 and MR3, which suggests that pharmacological modulation of these receptors can be used to raise the contractility of an inflamed uterus.

Central muscarinic receptor subtypes (M1 and M3) involved in carbacol-induced hypophagia in neonatal broiler chicken.

Aim: Food intake regulated by a complex of physiologic mechanisms in the nervous system. Muscarinergic system has an important role in the central regulation of appetite in mammals, but there is no information for Muscarinic receptors in avian. The purpose of this study was to examine the effects of intracerebroventricular injection of carbachol (cholinergic agonist), Telenzepine (M1 receptor antagonist), AF-DX116 (M2 receptor antagonist), 4-DAMP (M3 receptor antagonist), and PD102807 (M4 receptor antagonist) on feeding behavior in 3-h food-deprived (FD3) neonatal broiler chicken.Materials and Methods: In experiment 1, chicken intracerebroventricular injected with carbachol (125, 250, and 500 nmol). In experiment 2, birds intracerebroventricular injected with telenzepine (125, 250, and 500 nmol). In experiments 3-5, birds intracerebroventricular injected with AF-DX 116 (125, 250, and 500 nmol), 4-DAMP (125, 250, and 500 nmol), and PD102807 (125, 250, and 500 nmol), respectively. In experiment 6, broilers intracerebroventricular injected with carbacol (500 nmol), co-injection of telenzepine (125 nmol)+carbacol (500 nmol), and 4-DAMP (125 nmol)+carbacol (500 nmol). In experiment 7, injection procedure was carbacol (500 nmol), co-injection of AF-DX116 (125 nmol)+carbacol (500 nmol), and PD102807 (125 nmol)+carbacol (500 nmol). Then, food intake measured until 120 min after injection.Results: According to the data, carbachol (250 and 500 nmol) significantly decreased food intake in comparison with control group (P < 0.05). Intracerebroventricular injection of telenzepine (250 and 500 nmol) and 4-DAMP (250 and 500 nmol) significantly increased food intake (P < 0.05). In addition, carbacol-induced hypophagia was significantly attenuated by co-injection of telenzepine + carbacol (P < 0.05). Also, co-injection of 4-DAMP + carbacol decreased the effect of carbacol on food intake (P < 0.05). However, AF-DX116 and PD102807 had no effect on hypophagia induced by carbacol (P > 0.05).Conclusion: These results suggest, hypophagic effect of muscarinergic system is mediated via M1 and M3 receptors in neonatal chicken.

Muscarinic M1 and M2 receptor subtypes play opposite roles in LPS-induced septic shock.

BACKGROUND: To compare pharmacologic effects of pirenzepine and AF-DX116, a selective competitive antagonist for M1 and M2 subtype muscarinic cholinergic receptors (mAChRs), respectively, with atropine, a non-selective competitive antagonist for mAChRs, on Lipopolysaccharide (LPS). METHODS: Male C57BL/6 mice were used to establish models of LPS-induced experimental endotoxemia. Mice were intraperitoneally injected 10 min prior to LPS injection with control (saline), atropine, pirenzepine and AF-DX116, respectively. Overall survival time was estimated using Kaplan-Meier plots. Inflammatory cytokine tumor necrosis factor-α (TNF-α) was monitored at various intervals after LPS injection and individual reagent administration. Pathological alternations in lungs and liver were analyzed. RESULTS: Pirenzepine and atropine pretreatment improved survival rate of LPS-induced septic shock; in contrast, AF-DX116 accelerated death from sepsis. Moreover, TNF-α plasma level was decreased in response to pirenzepine or atropine, whereas increased in response to AF-DX116. Pirenzepine and atropine relieved whereas AF-DX116 accelerated LPS-induced pulmonary and hepatic injury. Pirenzepine reduced proportion of M1 subtype of macrophages, while AF-DX116 promoted polarization of macrophages to M1 subtype. Pirenzepine pretreatment reduced while AF-DX116 enhanced expression of SOCS3 at mRNA level. CONCLUSIONS: The administration of pirenzepine and atropine may have beneficial effects on septic shock.

Metabolic and endocrinal effects of N-desmethyl-olanzapine in mice with obesity: Implication for olanzapine-associated metabolic changes.

Clinical use of the antipsychotic drug olanzapine (OLA) is associated with metabolic side effects to variable degrees. N-desmethyl-olanzapine (DMO) is one major metabolite of OLA, but its potential involvement in the metabolic responses remains unclear. Here we examined whether DMO can directly impact the metabolic, endocrinal and inflammatory parameters under conditions of metabolic disturbance. DMO administration (2 mg/kg, i.g.) to high-fat diet induced obesity mice for 4 weeks induced a remarkable loss of body weight and fat mass. DMO improved insulin resistance and energy expenditure in mice, but had no significant effects on dyslipidemia or hepatic steatosis. Moreover, DMO induced morphological changes in the white adipose tissue, accompanied by reduced interleukin-1ß (IL-1ß) production and increased UCP1 expression. These findings demonstrate that DMO is devoid of the metabolic side effects commonly observed for OLA during obesity, which suggests that the N-desmethyl metabolism may function to regulate the metabolic responses to OLA.

Novel choline analog 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol produces sympathoinhibition, hypotension, and antihypertensive effects.

The search for new drugs remains an important focus for the safe and effective treatment of cardiovascular diseases. Previous evidence has shown that choline analogs can offer therapeutic benefit for cardiovascular complications. The current study investigates the effects of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032) on cardiovascular function and cholinergic-nitric oxide signaling. Synthesized LQFM032 (0.3, 0.6, or 1.2 mg/kg) was administered by intravenous and intracerebroventricular routes to evaluate the potential alteration of mean arterial pressure, heart rate, and renal sympathetic nerve activity of normotensive and hypertensive rats. Vascular function was further evaluated in isolated vessels, while pharmacological antagonists and computational studies of nitric oxide synthase and muscarinic receptors were performed to assess possible mechanisms of LQFM032 activity. The intravenous and intracerebroventricular administration of LQFM032 elicited a temporal reduction in mean arterial pressure, heart rate, and renal sympathetic nerve activity of rats. The cumulative addition of LQFM032 to isolated endothelium-intact aortic rings reduced vascular tension and elicited a concentration-dependent relaxation. Intravenous pretreatment with L-NAME (nitric oxide synthase inhibitor), atropine (nonselective muscarinic receptor antagonist), pirenzepine, and 4-DAMP (muscarinic M1 and M3 subtype receptor antagonist, respectively) attenuated the cardiovascular effects of LQFM032. These changes may be due to a direct regulation of muscarinic signaling as docking data shows an interaction of choline analog with M1 and M3 but not nitric oxide synthase. Together, these findings demonstrate sympathoinhibitory, hypotensive, and antihypertensive effects of LQFM032 and suggest the involvement of muscarinic receptors.

Effects of time-of-day on inhibition of lens-induced myopia by quinpirole, pirenzepine and atropine in chicks.

Injections of the D2 dopamine receptor agonist quinpirole or the acetylcholine muscarinic receptor antagonists pirenzepine and atropine prevent the development of negative-lens-induced myopia in chicks by inhibiting ocular growth. Because ocular growth is diurnally rhythmic, we hypothesized that the efficacy for inhibition may depend on time of day. Chicks wore monocular -10D lenses for 5 days, starting at 12d of age. The light cycle was 12L/12D. The lens-wearing eye received daily intravitreal injections for 4 days, of 20 µl quinpirole (10 nmol), at the following times: 7:30 EST (lights-on; morning; n = 12), 12:00 (mid-day; n = 13), or 19:30 (evening; n = 17). The same protocol was used for pirenzepine (0.2 µmol) and atropine (18 nmol), at the following times: 8:30 EDT (lights-on; n = 10; n = 18), 14:00 (n = 10; n = 12), or 20:30 (n = 18; n = 16). Saline injections were done in separate groups of birds for all groups as controls, and the data combined (n = 28). Ocular dimensions were measured using A-scan ultrasonography on treatment day 1 at 12:00, and again on day 5 at 12:00; growth rate is defined as the change in axial length over 96 h. For quinpirole and pirenzepine, subsets (n's in Methods) of mid-day and evening groups were measured at 6 h intervals on day 5 (from 12:00 to 12:00) to obtain rhythm parameters for axial length and choroidal thickness; for atropine, only the mid-day group was measured. Refractions were measured on day 5 with a Hartinger's refractometer. For quinpirole and pirenzepine, mid-day injections were more effective at inhibiting ocular growth than evening (Exp-fellow: quinpirole: -68 vs 118 µm/96h; post-hoc Bonferroni p = 0.016; pirenzepine: 79 vs 215 µm/96h; p = 0.046). There were no between-group statistically significant differences for atropine. For quinpirole, the mid-day amplitude of the axial rhythm was smaller than for evening (95 vs 142 µm; p < 0.05), but there were no time-dependent effects on the rhythms for pirenzepine. For atropine, the amplitude of the axial-length rhythm was significantly larger than that for pirenzepine at mid-day. We conclude that there is a phase-dependent efficacy for quinpirole and pirenzepine, with mid-day injections being most effective. There were no consistent time-dependent alterations in rhythm parameters for any of the drugs.

Contribution of M1 and M2 muscarinic receptor subtypes to convulsions in fasted mice treated with scopolamine and given food.

Treatment of fasted mice and rats with the nonselective muscarinic antagonist, scopolamine or atropine, causes convulsions after food intake. This study evaluated the effect of fasting on the expression of M1 and M2 muscarinic receptors in the brain regions, the relationship between receptor expression and seizure stages, and the muscarinic receptor subtype which plays a role in the occurrence of convulsions. Mice were grouped as allowed to eat ad lib (fed) and deprived of food for 24h (fasted). Fasted animals developed convulsions after being treated with scopolamine (60%) or the selective M1 receptor antagonist pirenzepine (10mg/kg; 20% and 60mg/kg; 70%) and given food. Fasting increased expression of M1 receptors in the frontal cortex and M2 receptors in the hippocampus, but produced no change in the expression of both receptors in the amygdaloid complex. Food intake after fasting decreased M1 receptor expression in the frontal cortex and M1 and M2 receptor expression in the hippocampus. Seizure severity was uncorrelated with muscarinic receptor expression in the brain regions. Taken together, these findings provide evidence for the role of M1 muscarinic receptor antagonism and fasting-induced increases in M1 and M2 expression possible underlying mechanism in the occurrence of convulsions in fasted animals.

Central muscarinic and LPBN mechanisms on sodium intake.

Central cholinergic activation stimulates water intake, but also NaCl intake when the inhibitory mechanisms are blocked with injections of moxonidine (α2 adrenergic/imidazoline agonist) into the lateral parabrachial nucleus (LPBN). In the present study, we investigated the involvement of central M1 and M2 muscarinic receptors on NaCl intake induced by pilocarpine (non-selective muscarinic agonist) intraperitoneally combined with moxonidine into the LPBN or by muscimol (GABAA agonist) into the LPBN. Male Holtzman rats with stainless steel cannulas implanted bilaterally in the LPBN and in the lateral ventricle were used. Pirenzepine (M1 muscarinic antagonist, 1 nmol/1 µl) or methoctramine (M2 muscarinic antagonist, 50 nmol/1 µL) injected intracerebroventricularly (i.c.v.) reduced 0.3 M NaCl and water intake in rats treated with pilocarpine (0.1 mg/100 g of body weight) injected intraperitoneally combined with moxonidine (0.5 nmol/0.2 µL) into the LPBN. In rats treated with muscimol (0.5 nmol/0.2 µL) into the LPBN, methoctramine i.c.v. also reduced 0.3 M NaCl and water intake, however, pirenzepine produced no effect. The results suggest that M1 and M2 muscarinic receptors activate central pathways involved in the control of water and sodium intake that are under the influence of the LPBN inhibitory mechanisms.

Striatal cholinergic interneurons regulate cognitive and affective dysfunction in partially dopamine-depleted mice.

Early non-motor symptoms such as mood disorders and cognitive deficits are increasingly recognised in Parkinson's disease (PD). They may precede the characteristic motor symptomatology caused by dopamine (DA) neuronal loss in the substantia nigra pars compacta (SNc). It is well known that striatal cholinergic interneurons (ChIs) are emerging as key regulators of PD motor symptom, however, their involvement in the cognitive and affective alterations occurring in the premotor phase of PD is poorly understood. We used optogenetic photoinhibition of striatal ChIs in mice with mild nigrostriatal 6-hydroxydopamine (6-OHDA) lesions and assessed their role in anxiety-like behaviour in the elevated plus maze, social memory recognition of a congener and visuospatial object recognition. In transgenic mice specifically expressing halorhodopsin (eNpHR) in cholinergic neurons, striatal ChIs photoinhibition reduced the anxiety-like behaviour and reversed social and spatial short-term memory impairment induced by moderate DA depletion (e.g., 50% loss of tyrosine hydroxylase TH-positive neurons in the SNc). Systemic injection of telenzepine (0.3 mg/kg), a preferential M1 muscarinic cholinergic receptors antagonist, improved anxiety-like behaviour, social memory recognition but not spatial memory deficits. Our results suggest that dysfunction of the striatal cholinergic system may play a role in the short-term cognitive and emotional deficits of partially DA-depleted mice. Blocking cholinergic activity with M1 muscarinic receptor antagonists may represent a possible therapeutic target, although not exclusive, to modulate these early non-motor deficits.