Increased cholinergic activity under conditions of low estrogen leads to adverse cardiac remodeling.

Cholinesterase inhibitors are used in postmenopausal women for the treatment of neurodegenerative diseases. Despite their widespread use in the clinical practice, little is known about the impact of augmented cholinergic signaling on cardiac function under reduced estrogen conditions. To address this gap, we subjected a genetically engineered murine model of systemic vesicular acetylcholine transporter overexpression (Chat-ChR2) to ovariectomy and evaluated cardiac parameters. Left-ventricular function was similar between Chat-ChR2 and wild-type (WT) mice. Following ovariectomy, WT mice showed signs of cardiac hypertrophy. Conversely, ovariectomized (OVX) Chat-ChR2 mice evolved to cardiac dilation and failure. Transcript levels for cardiac stress markers atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were similarly upregulated in WT/OVX and Chat-ChR2/OVX mice. 17ß-Estradiol (E2) treatment normalized cardiac parameters in Chat-ChR2/OVX to the Chat-ChR2/SHAM levels, providing a link between E2 status and the aggravated cardiac response in this model. To investigate the cellular basis underlying the cardiac alterations, ventricular myocytes were isolated and their cellular area and contractility were assessed. Myocytes from WT/OVX mice were wider than WT/SHAM, an indicative of concentric hypertrophy, but their fractional shortening was similar. Conversely, Chat-ChR2/OVX myocytes were elongated and presented contractile dysfunction. E2 treatment again prevented the structural and functional changes in Chat-ChR2/OVX myocytes. We conclude that hypercholinergic mice under reduced estrogen conditions do not develop concentric hypertrophy, a critical compensatory adaptation, evolving toward cardiac dilation and failure. This study emphasizes the importance of understanding the consequences of cholinesterase inhibition, used clinically to treat dementia, for cardiac function in postmenopausal women.

Age-related decrease of cholinergic parasympathetic reflex vasodilation in the rat masseter muscle.

Skeletal muscle hemodynamics, including that in jaw muscles, is an important in their functions and is modulated by aging. Marked blood flow increases mediated by parasympathetic vasodilation may be important for blood flow in the masseter muscle (MBF); however, the relationship between parasympathetic vasodilation and aging is unclear. We examined the effect of aging on parasympathetic vasodilation evoked by trigeminal afferent inputs and their mechanisms by investigating the MBF during stimulation of the lingual nerve (LN) in young and old urethane-anesthetized and vago-sympathectomized rats. Electrical stimulation of the central cut end of the LN elicited intensity- and frequency-dependent increases in MBF in young rats, while these increases were significantly reduced in old rats. Increases in the MBF evoked by LN stimulation in the young rats were greatly reduced by hexamethonium and atropine administration. Increases in MBF in young rats were produced by exogenous acetylcholine in a dose-dependent manner, whereas acetylcholine did not influence the MBF in old rats. Significant levels of muscarinic acetylcholine receptor type 1 (MR1) and type 3 (MR3) mRNA were observed in the masseter muscle in young rats, but not in old rats. Our results indicate that cholinergic parasympathetic reflex vasodilation evoked by trigeminal afferent inputs to the masseter muscle is reduced by aging and that this reduction may be mediated by suppression of the expression of MR1 and MR3 in the masseter muscle with age.

Early life voiding dysfunction leads to lower urinary tract dysfunction through alteration of muscarinic and purinergic signaling in the bladder.

Lower urinary tract dysfunction (LUTD) is a common problem in children and constitutes up to 40% of pediatric urology clinic visits. Improved diagnosis and interventions have been leading to better outcomes in many patients, whereas some children are left untreated or do not respond to the treatment successfully. In addition, many of these patients are lost by the pediatric urologists during their teenage years, and the outcome in later life largely remains unidentified. Studies suggest childhood LUTD is associated with subsequent adult urinary tract symptoms. However, whether and how early life LUTD attributes to urinary symptoms in those patients later in life remains to be elucidated. In the current study, we investigated the effects of early life voiding perturbation on bladder function using a neonatal maternal separation (NMS) protocol in mice. The NMS group displayed a delayed development of voluntary voiding behavior, a significant reduction of functional bladder capacity, and bladder overactivity compared with control mice later in life. In vitro evaluation of detrusor smooth muscle and molecular study showed a decrease in muscarinic contribution alongside an increase in purinergic contribution in detrusor contractility in NMS mice compared with control group. These results suggest that early life bladder dysfunction interfered with the normal maturation of the voluntary micturition control and facilitated LUTD in a later stage, which is at least partly attributed to an alteration of muscarinic and purinergic signaling in the urinary bladder.

BDNF overexpression in the bladder induces neuronal changes to mediate bladder overactivity.

Elevated levels of brain-derived neurotrophic factor (BDNF) in urine of overactive bladder (OAB) patients support the association of BDNF with OAB symptoms, but the causality is not known. Here, we investigated the functionality of BDNF overexpression in rat bladder following bladder wall transfection of either BDNF or luciferase (luciferase) transgenes (10 µg). One week after transfection, BDNF overexpression in bladder tissue and elevation of urine BDNF levels were observed together with increased transcript of BDNF, its cognate receptors (TrkB and p75NTR), and downstream PLCγ isoforms in bladder. BDNF overexpression can induce the bladder overactivity (BO) phenotype which is demonstrated by the increased voiding pressure and reduced intercontractile interval during transurethral open cystometry under urethane anesthesia. A role for BDNF-mediated enhancement of prejunctional cholinergic transmission in BO is supported by the significant increase in the atropine- and neostigmine-sensitive component of nerve-evoked contractions and upregulation of choline acetyltransferase, vesicular acetylcholine transporter, and transporter Oct2 and -α1 receptors. In addition, higher expression of transient receptor channels (TRPV1 and TRPA1) and pannexin-1 channels in conjunction with elevation of ATP and neurotrophins in bladder and also in L6/S1 dorsal root ganglia together support a role for sensitized afferent nerve terminals in BO. Overall, genomic changes in efferent and afferent neurons of bladder induced by the overexpression of BDNF per se establish a mechanistic link between elevated BDNF levels in urine and dysfunctional voiding observed in animal models and in OAB patients.

Apelin-13 inhibits gastric motility through vagal cholinergic pathway in rats.

The expression of apelin and its receptors (APJ) in central autonomic networks suggests that apelin may regulate gastrointestinal motor functions. In rodents, central administration of apelin-13 has been shown to inhibit gastric emptying; however, the mechanisms involved remain to be determined. Using male adult Sprague-Dawley rats, the aims of the present study were 1) to determine the expression of APJ receptor in the dorsal vagal complex (DVC), 2) to assess the effects of central application of apelin-13 into the DVC on gastric tone and motility, and 3) to investigate the neuronal pathways responsible for apelin-induced alterations. APJ receptor immunoreactivity was detected in gastric-projecting and choline acetyltransferase-positive neurons of the DVC. Microinjection of apelin-13 into the DVC significantly decreased gastric tone and motility in both corpus and antrum. The apelin-induced reduction in gastric tone and motility was prevented by surgical vagotomy or fourth ventricular application of the APJ receptor antagonist, [Ala13]apelin-13 (F13A). Systemic administration of the muscarinic receptor antagonist atropine, but not the nitric oxide synthase inhibitor nitro-l-arginine methyl ester (l-NAME), abolished the apelin-induced inhibitory responses. The present results indicate a central modulatory role of apelin in the vagal neurocircuitry that controls gastric motor functions via withdrawal of the tonically active cholinergic pathway. NEW & NOTEWORTHY This is the first study investigating the effects induced by brain stem application of apelin-13 while monitoring gastric tone and motility in rats. We have found that gastric-projecting neurons of the dorsal vagal complex express apelin receptors (APJ), which mediate the inhibitory actions of apelin-13. The inhibitory effects of apelin were abolished by systemic preadministration of atropine, but not nitro-l-arginine methyl ester (l-NAME). Apelin seems to modulate gastric motility via withdrawal of the tonically active vagal cholinergic pathway.

Inhibition of cholinergic neurotransmission by ß3-adrenoceptors depends on adenosine release and A1-receptor activation in human and rat urinary bladders.

The direct detrusor relaxant effect of ß3-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of ß3-adrenoceptors downmodulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A1 receptors to explain inhibition of cholinergic activity by ß3-adrenoceptors. Isoprenaline (1 µM) decreased [3H]ACh release from stimulated (10 Hz, 200 pulses) human (-47 ± 5%) and rat (-38 ± 1%) detrusor strips. Mirabegron (0.1 µM, -53 ± 8%) and CL316,243 (1 µM, -37 ± 7%) mimicked isoprenaline (1 µM) inhibition, and their effects were prevented by blocking ß3-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively, in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor, and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole, and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1,000 nM). Data suggest that inhibition of cholinergic neurotransmission by ß3-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A1-receptor stimulation in human and rat urinary bladder.

Interactions between glycopyrronium and indacaterol on cholinergic neurotransmission and contractile response in bovine trachealis.

BACKGROUND: Muscarinic-receptor antagonists and ß-adrenoceptor agonists are used, alone or in combination, as first-line treatment for chronic obstructive pulmonary disease. Both drugs decrease airway smooth muscle tone by post-junctional mechanisms but they may have opposing effects on pre-junctional acetylcholine (ACh)-release. METHODS: We studied the effects of the muscarinic-receptor antagonist glycopyrronium (GLY), the ß-adrenoceptor agonist indacaterol (IND) and their combination on electrically-induced ACh-release and contractile response in isolated bovine trachealis. Data were analyzed by paired t-test and analysis of variance for repeated or independent measures with Newmann-Keuls post-hoc test when appropriate. RESULTS: GLY 10-8 M decreased contractile response by 19 ± 6% (p = 0.010) without altering ACh-release. GLY 10-7 M and 10-6 M almost abolished contractile responses even if the ACh-release was increased by 27 ± 19% (p < 0.001) and 20 ± 8% (p = 0.004), respectively. IND 10-7 M had no significant effects on contractile response and ACh-release, whereas IND 10-6 M reduced contractile response by 24 ± 12% (p = 0.002) without altering ACh-release. IND 10-5 M decreased contractile response by 51 ± 17% (p < 0.001) and ACh-release by 22 ± 11% (p = 0.004). Co-incubation with GLY 10-8 M and IND 10-7 M did not alter ACh-release but inhibited contractile response by 41 ± 8% (p < 0.001). The latter effect was greater than with GLY 10-8 M, or IND 10-7 M, or IND 10-6 M given separately (p < 0.001 for all). The increment of ACh-release caused by GLY was attenuated by IND 10-5 M, though this did not affect contractile response. CONCLUSIONS: At equimolar concentration, GLY alone attenuates airway smooth muscle contraction more than IND, despite an increased ACh-release. Combination of GLY with IND at submaximal concentrations has more than additive effect suggesting a synergistic post-junctional effect. Adding GLY to IND provides a greater inhibitory effect on airway smooth muscle contraction than increasing IND concentration.

Beta-3 adrenergic receptor is expressed in acetylcholine-containing nerve fibers of the human urinary bladder: An immunohistochemical study.

AIMS: To identify in the human bladder the structures which express the Beta-3 adrenoceptor (ß3AR). METHODS: Human bladders from cadaveric organ donors (equally balanced in sex and age) were collected. Bladders were immediately fixed in paraformaldehyde and further processed for cryostat sectioning. Single and double immunohistochemistry was performed using antibodies against ß3AR C-terminal, ß3AR N-terminal, a pan-neuronal marker (ß3-Tubulin) and markers of cholinergic (Vesicular Acetylcholine Transporter), adrenergic (Tyrosine Hidroxylase), and peptidergic (Calcitonin Gene-Related Peptide) nerve fibers. RESULTS: Nerve fibers expressing immunoreactivity for ß3AR were abundantly found in the mucosa and muscular layers of the human bladder. No ß3AR-IR was detected on urothelial or smooth muscle cells. The presence of ß3AR-IR in nerve fibers was confirmed by co-expression with ß3-Tubulin. Nerve fibers expressing ß3AR-IR were cholinergic, VAChT+ , and abundantly observed in the suburothelium. The cholinergic fibers were in close proximity and intermingled with adrenergic TH+ and peptidergic CGRP+ fibers. CONCLUSIONS: We demonstrated that ß3AR is abundantly located in acetylcholine-containing nerve fibers. These findings have important consequences to understand the mechanism of action of ß3AR agonists currently used for the treatment of OAB.

Novel mechanism of hydrogen sulfide-induced guinea pig urinary bladder smooth muscle contraction: role of BK channels and cholinergic neurotransmission.

Hydrogen sulfide (H2S) is a key signaling molecule regulating important physiological processes, including smooth muscle function. However, the mechanisms underlying H2S-induced detrusor smooth muscle (DSM) contractions are not well understood. This study investigates the cellular and tissue mechanisms by which H2S regulates DSM contractility, excitatory neurotransmission, and large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in freshly isolated guinea pig DSM. We used a multidisciplinary experimental approach including isometric DSM tension recordings, colorimetric ACh measurement, Ca(2+) imaging, and patch-clamp electrophysiology. In isolated DSM strips, the novel slow release H2S donor, P-(4-methoxyphenyl)-p-4-morpholinylphosphinodithioic acid morpholine salt (GYY4137), significantly increased the spontaneous phasic and nerve-evoked DSM contractions. The blockade of neuronal voltage-gated Na(+) channels or muscarinic ACh receptors with tetrodotoxin or atropine, respectively, reduced the stimulatory effect of GYY4137 on DSM contractility. GYY4137 increased ACh release from bladder nerves, which was inhibited upon blockade of L-type voltage-gated Ca(2+) channels with nifedipine. Furthermore, GYY4137 increased the amplitude of the Ca(2+) transients and basal Ca(2+) levels in isolated DSM strips. GYY4137 reduced the DSM relaxation induced by the BK channel opener, NS11021. In freshly isolated DSM cells, GYY4137 decreased the amplitude and frequency of transient BK currents recorded in a perforated whole cell configuration and reduced the single BK channel open probability measured in excised inside-out patches. GYY4137 inhibited spontaneous transient hyperpolarizations and depolarized the DSM cell membrane potential. Our results reveal the novel findings that H2S increases spontaneous phasic and nerve-evoked DSM contractions by activating ACh release from bladder nerves in combination with a direct inhibition of DSM BK channels.

Signaling pathways relevant to cognition-enhancing drug targets.

Aging is generally associated with a certain cognitive decline. However, individual differences exist. While age-related memory deficits can be observed in humans and rodents in the absence of pathological conditions, some individuals maintain intact cognitive functions up to an advanced age. The mechanisms underlying learning and memory processes involve the recruitment of multiple signaling pathways and gene expression, leading to adaptative neuronal plasticity and long-lasting changes in brain circuitry. This chapter summarizes the current understanding of how these signaling cascades could be modulated by cognition-enhancing agents favoring memory formation and successful aging. It focuses on data obtained in rodents, particularly in the rat as it is the most common animal model studied in this field. First, we will discuss the role of the excitatory neurotransmitter glutamate and its receptors, downstream signaling effectors [e.g., calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), extracellular signal-regulated kinases (ERK), mammalian target of rapamycin (mTOR), cAMP response element-binding protein (CREB)], associated immediate early gene (e.g., Homer 1a, Arc and Zif268), and growth factors [insulin-like growth factors (IGFs) and brain-derived neurotrophic factor (BDNF)] in synaptic plasticity and memory formation. Second, the impact of the cholinergic system and related modulators on memory will be briefly reviewed. Finally, since dynorphin neuropeptides have recently been associated with memory impairments in aging, it is proposed as an attractive target to develop novel cognition-enhancing agents.

Cholinergic immunotoxin 192 IgG-SAPORIN alters subicular theta-gamma activity and impairs spatial learning in rats.

Subiculum is an important structure of hippocampal formation and is a part of intra hippocampal network involved in spatial information processing. However, relatively very few studies are available in literature demonstrating the explicit role of subiculum in spatial information processing. The present study investigated the cholinergic modulation of subicular theta-gamma activity on spatial learning and memory functions in rats. The cholinergic projections to ventral subiculum were selectively eliminated using 192 IgG-SAPORIN. Eliminations of cholinergic inputs to ventral subiculum significantly reduced the subicular theta and enhanced the gamma activity during active wake and REM sleep states. In addition, the spatial learning was severely impaired following cholinergic elimination of ventral subiculum. The ChAT immunocytochemical studies showed sparse distribution of cholinergic fibers in the ventral subiculum confirming the cholinergic elimination to ventral subiculum. Cholinotoxic infusions to ventral subiculum did not alter the hippocampal cholinergic innervations and retained the hippocampal theta and gamma activities. The present findings support that cholinergic modulation of subicular theta-gamma oscillations is crucial for spatial information processing.

Enhanced striatal cholinergic neuronal activity mediates L-DOPA-induced dyskinesia in parkinsonian mice.

Treatment of Parkinson disease (PD) with L-3,4-dihydroxyphenylalanine (L-DOPA) dramatically relieves associated motor deficits, but L-DOPA-induced dyskinesias (LID) limit the therapeutic benefit over time. Previous investigations have noted changes in striatal medium spiny neurons, including abnormal activation of extracellular signal-regulated kinase1/2 (ERK). Using two PD models, the traditional 6-hydroxydopamine toxic lesion and a genetic model with nigrostriatal dopaminergic deficits, we found that acute dopamine challenge induces ERK activation in medium spiny neurons in denervated striatum. After repeated L-DOPA treatment, however, ERK activation diminishes in medium spiny neurons and increases in striatal cholinergic interneurons. ERK activation leads to enhanced basal firing rate and stronger excitatory responses to dopamine in striatal cholinergic neurons. Pharmacological blockers of ERK activation inhibit L-DOPA-induced changes in ERK phosphorylation, neuronal excitability, and the behavioral manifestation of LID. In addition, a muscarinic receptor antagonist reduces LID. These data indicate that increased dopamine sensitivity of striatal cholinergic neurons contributes to the expression of LID, which suggests novel therapeutic targets for LID.

Cholinergic markers in the cortex and hippocampus of some animal species and their correlation to Alzheimer's disease.

INTRODUCTION: The cholinergic system includes neurons located in the basal forebrain and their long axons that reach the cerebral cortex and the hippocampus. This system modulates cognitive function. In Alzheimer's disease (AD) and ageing, cognitive impairment is associated with progressive damage to cholinergic fibres, which leads us to the cholinergic hypothesis for AD. DEVELOPMENT: The AD produces alterations in the expression and activity of acetyltransferase (ChAT) and acetyl cholinesterase (AChE), enzymes specifically related to cholinergic system function. Both proteins play a role in cholinergic transmission, which is altered in both the cerebral cortex and the hippocampus due to ageing and AD. Dementia disorders are associated with the severe destruction and disorganisation of the cholinergic projections extending to both structures. Specific markers, such as anti-ChAT and anti-AChE antibodies, have been used in light immunohistochemistry and electron microscopy assays to study this system in adult members of certain animal species. CONCLUSIONS: This paper reviews the main immunomorphological studies of the cerebral cortex and hippocampus in some animal species with particular emphasis on the cholinergic system and its relationship with the AD.

SPARC prevents maturation of cholinergic presynaptic terminals.

Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein produced by glial cells. Although it is highly expressed in synaptogenic areas in the developing nervous system, it is still unclear whether this molecule displays an action on synaptic activity. We show that nanomolar concentrations of SPARC favour a more efficient synapse formation and increase short term depression in single cell cholinergic microcultures. The change in synaptic plasticity, which is also observed when SPARC is locally secreted on stable synapses for 24-48 h, is caused by a high release probability and a reduction in the size of the rapidly releasable pool of vesicles. Both features are attributable to synapses operating at an immature stage as demonstrated by correlative electrophysiology and electron microscopy experiments. Presynaptic terminals developed in the presence of SPARC display few cytoplasmic vesicles and two to threefold decrease in the number of docked vesicles at active zones. At the postsynaptic level, the analysis of miniature excitatory postsynaptic currents suggests SPARC has little effect on the number of nicotinic receptors but might alter their composition. The widespread distribution of SPARC makes current findings potentially relevant to other excitatory synapses and development of neuronal circuits.

Physiological activation of cholinergic inputs controls associative synaptic plasticity via modulation of endocannabinoid signaling.

Cholinergic neuromodulation controls long-term synaptic plasticity underlying memory, learning, and adaptive sensory processing. However, the mechanistic interaction of cholinergic, neuromodulatory inputs with signaling pathways underlying long-term potentiation (LTP) and long-term depression (LTD) remains poorly understood. Here, we show that physiological activation of muscarinic acetylcholine receptors (mAChRs) controls the size and sign of associative long-term synaptic plasticity via interaction with endocannabinoid signaling. Our findings indicate that synaptic or pharmacological activation of postsynaptic M1/M3 converts postsynaptic Hebbian LTP to presynaptic anti-Hebbian LTD in principal neurons of the dorsal cochlear nucleus (DCN). This conversion is also dependent on NMDA receptor (NMDAR) activation and rises in postsynaptic Ca(2+). While NMDAR activation and Ca(2+) elevation lead to LTP, when these events are coordinated with simultaneous activation of M1/M3 mAChRs, anti-Hebbian LTD is induced. Anti-Hebbian LTD is mediated by a postsynaptic G-protein-coupled receptor intracellular signaling cascade that activates phospholipase C and that leads to enhanced endocannabinoid signaling. Moreover, the interaction between postsynaptic M1/M3 mAChRs and endocannabinoid signaling is input specific, as it occurs only in the parallel fiber inputs, but not in the auditory nerve inputs innervating the same DCN principal neurons. Based on the extensive distribution of cholinergic and endocannabinoid signaling, we suggest that their interaction may provide a general mechanism for dynamic, context-dependent modulation of associative synaptic plasticity.

Neural signaling in the spleen controls B-cell responses to blood-borne antigen.

Entry of blood-borne pathogens into the spleen elicits a series of changes in cellular architecture that culminates in the systemic release of protective antibodies. Despite an abundance of work that has characterized these processes, the regulatory mechanisms that coordinate cell trafficking and antibody production are still poorly understood. Here, marginal zone (MZ) B cells responding to streptococcus in the blood were observed to migrate along splenic nerves, arriving at the red pulp venous sinuses where they become antibody-secreting cells. Electrical stimulation of the vagus nerve, which in turn regulates the splenic nerve, arrested B-cell migration and decreased antibody secretion. Thus, neural circuits regulate the first wave of antibody production following B-cell exposure to blood-borne antigen.

A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease.

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.

Extracts of Cordyceps militaris lower blood glucose via the stimulation of cholinergic activation and insulin secretion in normal rats.

Previous studies have shown that Cordyceps militaris (CM) has a hypoglycemic effect, but the actual mechanism remains unclear. This study explored the hypoglycemic mechanism of aqueous extracts of CM in normal Wistar rats. First, the optimal dose of CM for lowering plasma glucose and insulin secretion was tested. Further, atropine and hemicholinium-3 (HC-3) were injected and a western blot was used to investigate insulin signaling. It was found that 10 mg/kg CM extracts had a stronger hypoglycemic effect than a higher dose (100 mg/kg); therefore, a dose of 10 mg/kg was used in subsequent experiments. In normal rats, CM extracts decreased plasma glucose by 21.0% and induced additional insulin secretion by 54.5% after 30 min. When atropine or HC-3 was injected, CM induced a hypoglycemic effect, but the enhancement of insulin secretion was blocked. By western blotting, significant increases in the insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT-4) were observed after CM feeding. However, the elevation of these signaling proteins was abolished by atropine or HC-3. Taken together, these findings indicate that CM can lower plasma glucose via the stimulation of insulin secretion and cholinergic activation involved in the hypoglycemic mechanism of normal Wistar rats.

The organization of cholinergic projections in the visual thalamus of the mouse.

Cholinergic projections from the brainstem serve as important modulators of activity in visual thalamic nuclei such as the dorsal lateral geniculate nucleus (dLGN). While these projections have been studied in several mammals, a comprehensive examination of their organization in the mouse is lacking. We used the retrograde transport of viruses or cholera toxin subunit B (CTB) injected in the dLGN, immunocytochemical labeling with antibodies against choline acetyltransferase (ChAT), brain nitric oxide synthase (BNOS), and vesicular acetylcholine transporter (VAChT), ChAT-Cre mice crossed with a reporter line (Ai9), as well as brainstem virus injections in ChAT-Cre mice to examine the pattern of thalamic innervation from cholinergic neurons in the pedunculopontine tegmental nucleus (PPTg), laterodorsal tegmental nucleus (LDTg), and the parabigeminal nucleus (PBG). Retrograde tracing demonstrated that the dLGN receives input from the PPTg, LDTg, and PBG. Viral tracing in ChAT-Cre mice and retrograde tracing combined with immunocytochemistry revealed that many of these inputs originate from cholinergic neurons in the PBG and PPTg. Most notable was an extensive cholinergic projection from the PBG which innervated most of the contralateral dLGN, with an especially dense concentration in the dorsolateral shell, as well as a small region in the dorsomedial pole of the ipsilateral dLGN. The PPTg was found to provide a sparse somewhat diffuse innervation of the ipsilateral dLGN. Neurons in the PPTg co-expressed ChAT, BNOS, and VAChT, whereas PBG neurons expressed ChAT, but not BNOS or VAChT. These results highlight the presence of distinct cholinergic populations that innervate the mouse dLGN.

Synaptic-like axo-axonal transmission from striatal cholinergic interneurons onto dopaminergic fibers.

Transmission from striatal cholinergic interneurons (CINs) controls dopamine release through nicotinic acetylcholine receptors (nAChRs) on dopaminergic axons. Anatomical studies suggest that cholinergic terminals signal predominantly through non-synaptic volume transmission. However, the influence of cholinergic transmission on electrical signaling in axons remains unclear. We examined axo-axonal transmission from CINs onto dopaminergic axons using perforated-patch recordings, which revealed rapid spontaneous EPSPs with properties characteristic of fast synapses. Pharmacology showed that axonal EPSPs (axEPSPs) were mediated primarily by high-affinity α6-containing receptors. Remarkably, axEPSPs triggered spontaneous action potentials, suggesting that these axons perform integration to convert synaptic input into spiking, a function associated with somatodendritic compartments. We investigated the cross-species validity of cholinergic axo-axonal transmission by recording dopaminergic axons in macaque putamen and found similar axEPSPs. Thus, we reveal that synaptic-like neurotransmission underlies cholinergic signaling onto dopaminergic axons, supporting the idea that striatal dopamine release can occur independently of somatic firing to provide distinct signaling.