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.

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.

Basal forebrain magnocellular cholinergic systems are damaged in mice following neonatal hypoxia-ischemia.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes lifelong neurologic disability. Despite the use of therapeutic hypothermia, memory deficits and executive functions remain severely affected. Cholinergic neurotransmission from the basal forebrain to neocortex and hippocampus is central to higher cortical functions. We examined the basal forebrain by light microscopy and reported loss of choline acetyltransferase-positive (ChAT)+ neurons, at postnatal day (P) 40, in the ipsilateral medial septal nucleus (MSN) after neonatal hypoxia-ischemia (HI) in mice. There was no loss of ChAT+ neurons in the ipsilateral nucleus basalis of Meynert (nbM) and striatum. Ipsilateral striatal and nbM ChAT+ neurons were abnormal with altered immunoreactivity for ChAT, shrunken and crenated somas, and dysmorphic appearing dendrites. Using confocal images with 3D reconstruction, nbM ChAT+ dendrites in HI mice were shorter than sham (p = .0001). Loss of ChAT+ neurons in the MSN directly correlated with loss of ipsilateral hippocampal area. In the nbM and striatum, percentage of abnormal ChAT+ neurons correlated with loss of ipsilateral cerebral cortical and striatal area, respectively. Acetylcholinesterase (AChE) activity increased in adjacent ipsilateral cerebral cortex and hippocampus and the increase was linearly related to loss of cortical and hippocampal area. Numbers and size of cathepsin D+ lysosomes increased in large neurons in the ipsilateral nbM. After neonatal HI, abnormalities were found throughout the major cholinergic systems in relationship to amount of forebrain area loss. There was also an upregulation of cathepsin D+ particles within the nbM. Cholinergic neuropathology may underlie the permanent dysfunction in learning, memory, and executive function after neonatal brain injury.

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.

Acetylcholine and spinal locomotor networks: The insider.

This article aims to review studies that have investigated the role of neurons that use the transmitter acetylcholine (ACh) in controlling the operation of locomotor neural networks within the spinal cord. This cholinergic system has the particularity of being completely intraspinal. We describe the different effects exerted by spinal cholinergic neurons on locomotor circuitry by the pharmacological activation or blockade of this propriospinal system, as well as describing its different cellular and subcellular targets. Through the activation of one ionotropic receptor, the nicotinic receptor, and five metabotropic receptors, the M1 to M5 muscarinic receptors, the cholinergic system exerts a powerful control both on synaptic transmission and locomotor network neuron excitability. Although tremendous advances have been made in our understanding of the spinal cholinergic system's involvement in the physiology and pathophysiology of locomotor networks, gaps still remain, including the precise role of the different subtypes of cholinergic neurons as well as their pre- and postsynaptic partners. Improving our knowledge of the propriospinal cholinergic system is of major relevance to finding new cellular targets and therapeutics in countering the debilitating effects of neurodegenerative diseases and restoring motor functions after spinal cord injury.

Loss of cholinergic innervation differentially affects eNOS-mediated blood flow, drainage of Aß and cerebral amyloid angiopathy in the cortex and hippocampus of adult mice.

Vascular dysregulation and cholinergic basal forebrain degeneration are both early pathological events in the development of Alzheimer's disease (AD). Acetylcholine contributes to localised arterial dilatation and increased cerebral blood flow (CBF) during neurovascular coupling via activation of endothelial nitric oxide synthase (eNOS). Decreased vascular reactivity is suggested to contribute to impaired clearance of ß-amyloid (Aß) along intramural periarterial drainage (IPAD) pathways of the brain, leading to the development of cerebral amyloid angiopathy (CAA). However, the possible relationship between loss of cholinergic innervation, impaired vasoreactivity and reduced clearance of Aß from the brain has not been previously investigated. In the present study, intracerebroventricular administration of mu-saporin resulted in significant death of cholinergic neurons and fibres in the medial septum, cortex and hippocampus of C57BL/6 mice. Arterial spin labelling MRI revealed a loss of CBF response to stimulation of eNOS by the Rho-kinase inhibitor fasudil hydrochloride in the cortex of denervated mice. By contrast, the hippocampus remained responsive to drug treatment, in association with altered eNOS expression. Fasudil hydrochloride significantly increased IPAD in the hippocampus of both control and saporin-treated mice, while increased clearance from the cortex was only observed in control animals. Administration of mu-saporin in the TetOAPPSweInd mouse model of AD was associated with a significant and selective increase in Aß40-positive CAA. These findings support the importance of the interrelationship between cholinergic innervation and vascular function in the aetiology and/or progression of CAA and suggest that combined eNOS/cholinergic therapies may improve the efficiency of Aß removal from the brain and reduce its deposition as CAA.

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.

Nucleus basalis of Meynert modulates signal processing in rat layer 5 somatosensory cortex but leads to memory impairment and tactile discrimination deficits following lesion.

In rodents, exploring through continuous whisking is a process resulted from sensorimotor networking among different layers of somatosensory cortex (SC) such as layer 5 (L5) or barrel field, and regions like the nucleus basalis of Meynert (NBM). NBM is densely packed with cholinergic fibers and its dysfunction leads to diminished acetylcholine release within SC, tactile deficits and Alzheimer's disease (AD)-like memory impairment. Using extracellular single-unit recording, we investigated mechanisms underlying changes in response characteristics of L5b neurons to single or paired deflection of selected principle and adjacent whiskers (PW and AW), following NBM electrical stimulation in normal rats or ibotenic acid-induced NBM lesion leading to potential tactile deficiency and memory loss during passive avoidance learning (PAL) in AD-like neuropathology. Our results indicated that NBM electrical stimulation decreased ON and OFF response magnitude in nearly half of the units upon vibrissal deflection. The larger the response was evoked to whisker deflection before NBM stimulation, the smaller it gets after stimulation. Neuronal spontaneous activity was not changed with NBM stimulation or lesion. Leading to more sublinear response summation and decreased condition-test ratio, NBM lesion decreased ON response magnitude and facilitation, increased AW surround inhibition in paired whisker deflection, increased excitatory and decreased inhibitory receptive fields, weakened information processing during whisking, and resulted in AD-like declined PAL performance. These findings provide further understandings to develop translational approaches in precision therapeutics to target highly specific regions such as NBM or SC, and pathways like cholinergic system involved in tactile and memory deficits in AD.

Distribution characteristics of sweat gland nerve fibres in normal humans identified by acetylcholinesterase histochemical staining.

OBJECTIVE: To quantitatively analyze distribution characteristics of sweat gland nerve fibres (SGNF) in normal Chinese individuals for obtaining a reference for early diagnosis of peripheral neuropathy. PATIENTS AND METHODS: Skin biopsy samples were collected from 192 normal Chinese individuals and divided into six, four and two groups according to anatomic sites, age and gender, respectively. SGNF morphology was observed and SGNF density (SGNFD) was determined. RESULTS: There was a significant difference in SGNFD among different anatomic sites, age and gender. A degressive tendency was observed from proximal to distal anatomic sites. SGNFD was the lowest in subjects in the 21-40-year-old age group, but was the highest in subjects in the >61-year-old age group. Overall, SGNFD fluctuated with age. SGNFD in males was significantly higher than that in females. CONCLUSIONS: Distribution characteristics of SGNF in normal individuals may serve as a reference for early diagnosis of nerve fibre damage.

The differential impact of acute microglia activation on the excitability of cholinergic neurons in the mouse medial septum.

The medial septal nucleus is one of the basal forebrain nuclei that projects cholinergic input to the hippocampus and cortex. Two of the hallmarks of Alzheimer's disease (AD) are a significant loss of cholinergic transmission and neuroinflammation, and it has been suggested that these two hallmarks are causally linked to the medial septum. Therefore, we have investigated the age-related susceptibility of medial septal cholinergic neurons to glial activation, mediated via peripheral administration of lipopolysaccharide (500 µg/kg) into ChAT(BAC)-eGFP mice at different ages (3-22 months). Our results show that during normal aging, cholinergic neurons experience a bi-phasic excitability profile, in which increased excitability at adulthood (ages ranging between 9 and 12 months) decreases in aged animals (> 18 months). Moreover, activation of glia had a differential impact on mice from different age groups, affecting K+ conductances in young and adult animals, without affecting aged mice. These findings provide a potential explanation for the increased vulnerability of cholinergic neurons to neuroinflammation with aging as reported previously, thus providing a link to the impact of acute neuroinflammation in AD.

Elaeagnus glabra f. oxyphylla Attenuates Scopolamine-Induced Learning and Memory Impairments in Mice by Improving Cholinergic Transmission via Activation of CREB/NGF Signaling.

We aimed to investigate the therapeutic effects of an Elaeagnus glabra f. oxyphylla (EGFO) ethanol extract in mice with scopolamine-induced memory dysfunction. Fifty male mice were randomly divided into a normal control group, a scopolamine-treated group, a scopolamine and EGFO extract-treated group, and a scopolamine and tacrine-treated group. EGFO (50 or 100 mg/kg/day) was received for 21 days. Step-through passive avoidance and Y-maze tests were performed to examine the effects of treatment on learning and memory impairments. Acetylcholine (Ach) levels and acetylcholinesterase (AchE) activity were measured via an enzyme-linked immunosorbent assay (ELISA). Levels of choline acetyltransferase (ChAT), nerve growth factor (NGF), cAMP response element-binding protein (CREB), and apoptosis-related protein expression were determined via Western blot analysis. EGFO pretreatment significantly attenuated scopolamine-induced memory impairments, relative to findings observed in the scopolamine-treated group. Levels of cholinergic factors in the brain tissues were markedly attenuated in the scopolamine-treated group. EGFO treatment also attenuated neural apoptosis in scopolamine-treated mice by decreasing the expression of apoptosis-related proteins such as Bax, Bcl2, cleaved caspase-3, and TUNEL staining. These results suggest that EGFO improves memory and cognition in a mouse model of memory impairment by restoring cholinergic and anti-apoptotic activity, possibly via activation of CREB/NGF signaling.

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.

Three-dimensional reconstructed eccrine sweat glands with vascularization and cholinergic and adrenergic innervation.

Functional integrity of the regenerated tissues requires not only structural integrity but also vascularization and innervation. We previously demonstrated that the three-dimensional (3D) reconstructed eccrine sweat glands had similar structures as those of the native ones did, but whether the 3D reconstructed glands possessing vascularization and innervation was still unknown. In the study, Matrigel-embedded eccrine sweat gland cells were implanted under the inguinal skin. Ten weeks post-implantation, the vascularization, and innervation in the 10-week reconstructed eccrine sweat glands and native human eccrine sweat glands were detected by immunofluorescence staining. The results showed that the fluorescent signals of general neuronal marker protein gene product 9.5, adrenergic nerve fiber marker tyrosine hydroxylase, and cholinergic nerve fiber markers acetylcholinesterase and vasoactive intestinal peptide embraced the 3D reconstructed glands in circular patterns, as the signals appeared in native eccrine sweat glands. There were many CD31- and von Willebrand factor-positive vessels growing into the plugs. We demonstrated that the 3D reconstructed eccrine sweat glands were nourished by blood vessels, and we for the first time demonstrated that the engineering sweat glands were innervated by both cholinergic and adrenergic fibers. In conclusion, the 3D reconstructed eccrine sweat glands may have functions as the native ones do.

The pedunculopontine and laterodorsal tegmental nuclei in the kainate model of epilepsy.

Prior studies showed that epilepsy can be associated with reorganization of the septohippocampal cholinergic fiber system. Using the kainate model of epilepsy, we wished to further examine the structural integrity of the mesopontine tegmental nuclei (pedunculopontine, PPN, and laterodorsal, LDT), which provide the cholinergic input to the thalamus. It was found that the total numbers of the PPN and LDT cells immunoreactive to the vesicular acetylcholine transporter did not differ between control and epileptic rats. However, the cholinergic cells had enlarged perikarya in epileptic rats. We further examined the effects of epilepsy on the distribution pattern of cholinergic fiber varicosities in the parafascicular nucleus, one of the principal thalamic targets of PPN projections. The density of cholinergic varicosities, represented by two distinct populations, was increased in epileptic rats. These data provide the first morphological evidence for structural alterations in mesopontine cholinergic neurons in experimental epilepsy. They suggest dysfunctional cholinergic transmission in the brainstem-thalamic pathway, which may partly account for various epilepsy-related neurological disturbances.

Variable expression of GFP in different populations of peripheral cholinergic neurons of ChATBAC-eGFP transgenic mice.

Immunohistochemistry is used widely to identify cholinergic neurons, but this approach has some limitations. To address these problems, investigators developed transgenic mice that express enhanced green fluorescent protein (GFP) directed by the promoter for choline acetyltransferase (ChAT), the acetylcholine synthetic enzyme. Although, it was reported that these mice express GFP in all cholinergic neurons and non-neuronal cholinergic cells, we could not detect GFP in cardiac cholinergic nerves in preliminary experiments. Our goals for this study were to confirm our initial observation and perform a qualitative screen of other representative autonomic structures for the presences of GFP in cholinergic innervation of effector tissues. We evaluated GFP fluorescence of intact, unfixed tissues and the cellular localization of GFP and vesicular acetylcholine transporter (VAChT), a specific cholinergic marker, in tissue sections and intestinal whole mounts. Our experiments identified two major tissues where cholinergic neurons and/or nerve fibers lacked GFP: 1) most cholinergic neurons of the intrinsic cardiac ganglia and all cholinergic nerve fibers in the heart and 2) most cholinergic nerve fibers innervating airway smooth muscle. Most cholinergic neurons in airway ganglia stained for GFP. Cholinergic systems in the bladder and intestines were fully delineated by GFP staining. GFP labeling of input to ganglia with long preganglionic projections (vagal) was sparse or weak, while that to ganglia with short preganglionic projections (spinal) was strong. Total absence of GFP might be due to splicing out of the GFP gene. Lack of GFP in nerve projections from GFP-positive cell bodies might reflect a transport deficiency.

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.

Developmental suppression of forebrain trkA receptors and attentional capacities in aging rats: A longitudinal study.

Basal forebrain (BF) cholinergic neurons innervating the cortex regulate cognitive, specifically attentional, processes. Cholinergic atrophy and cognitive decline occur at an accelerated pace in age-related neurodegenerative disorders such as Alzheimer's disease; however, the mechanism responsible for this phenomenon remains unknown. Here we hypothesized that developmental suppression of nerve growth factor signaling, mediated via tropomyosin-related kinase A (trkA) receptors, would escalate age-related attentional vulnerability. An adeno-associated viral vector expressing trkA shRNA (AAV-trkA) was utilized to knockdown trkA receptors in postnatal rats at an ontogenetic time point when cortical cholinergic inputs mature, and the impact of this manipulation on performance was assessed in animals maintained on an operant attention task throughout adulthood and until old (24 months) age. A within-subject comparison across different time points illustrated a gradual age-related decline in attentional capacities. However, the performance under baseline and distracted conditions did not differ between the AAV-trkA-infused and animals infused with a vector expressing shRNA against the control protein luciferase at any time point. Additional analysis of cholinergic measures conducted at 24 months showed that the capacity of cholinergic terminals to release acetylcholine following a depolarizing stimulus, cortical cholinergic fiber density and BF cholinergic cell size remained comparable between the two groups. Contrary to our predictions, these data indicate that developmental BF trkA disruption does not impact age-related changes in attentional functions. It is possible that life-long engagement in cognitive activity might have potentially rescued the developmental insults on the cholinergic system, thus preserving attentional capacities in advanced age.

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.

Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress.

We have previously demonstrated that maternal exposure to glyphosate-based herbicide (GBH) leads to glutamate excitotoxicity in 15-day-old rat hippocampus. The present study was conducted in order to investigate the effects of subchronic exposure to GBH on some neurochemical and behavioral parameters in immature and adult offspring. Rats were exposed to 1% GBH in drinking water (corresponding to 0.36% of glyphosate) from gestational day 5 until postnatal day (PND)-15 or PND60. Results showed that GBH exposure during both prenatal and postnatal periods causes oxidative stress, affects cholinergic and glutamatergic neurotransmission in offspring hippocampus from immature and adult rats. The subchronic exposure to the pesticide decreased L-[14C]-glutamate uptake and increased 45Ca2+ influx in 60-day-old rat hippocampus, suggesting a persistent glutamate excitotoxicity from developmental period (PND15) to adulthood (PND60). Moreover, GBH exposure alters the serum levels of the astrocytic protein S100B. The effects of GBH exposure were associated with oxidative stress and depressive-like behavior in offspring on PND60, as demonstrated by the prolonged immobility time and decreased time of climbing observed in forced swimming test. The mechanisms underlying the GBH-induced neurotoxicity involve the NMDA receptor activation, impairment of cholinergic transmission, astrocyte dysfunction, ERK1/2 overactivation, decreased p65 NF-κB phosphorylation, which are associated with oxidative stress and glutamate excitotoxicity. These neurochemical events may contribute, at least in part, to the depressive-like behavior observed in adult offspring.