Corneal acetylcholine regulates sensory nerve activity via nicotinic receptors.

PURPOSE: Sensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea. METHODS: Localization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [3H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs. RESULTS: ChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [3H] acetylcholine was synthesized from [3H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist. CONCLUSIONS: The corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation.

An improved synthesis of [18 F]VAT and its precursor.

The vesicular acetylcholine transporter (VAChT) in the brain is an important presynaptic cholinergic biomarker, and neuroimaging studies of VAChT may provide in vivo information about psychiatric and neurologic conditions including Alzheimer's disease that are not accessible by other methods. The 18 F-labeled radiotracer, ((-)-(1-(-8-(2-[18 F]fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)-methanone ([18 F]VAT, 1), was reported as a selective and high affinity ligand for the in vivo imaging of VAChT. The synthesis of [18 F]VAT has been reported in a two-step procedure with total 140 min, which includes preparation of 2-[18 F]fluoroethyltosylate and alkylation of benzovesamicol (-)-5 precursor with this radiosynthon using two different automated production modules consecutively. A multiple step synthetic route was employed for the synthesis of stereospecific precursor benzovesamicol (-)-5, which is difficult to be adapted for scale-up. To make the production of this tracer more amenable for clinical imaging, we present an improved total synthesis protocol to attain [18 F]VAT: (1) a tosylethoxy group being pre-installed tosylate precursor (-)-8 is synthesized to render a simple one-step radiofluorination under mild conditions; (2) The key optically active intermediate benzovesamicol (-)-5 was obtained via the regio- and enantio-enriched ring-opening amination of meso-epoxide 3 with 4-phenylpiperidine derivative 2 under catalysis of a chiral salenCo(III) catalyst 4b, which dramatically simplifies the synthetic route of the tosylate precursor (-)-8. [18 F]VAT 1 was prepared within ~65 min with desired chemical and radiochemical purities, via a fully automated procedure, using a commercial PET tracer production module. The final drug product was obtained as a sterile, pyrogen-free solution that conforms United States Pharmacopeia (USP) <823> requirements.

Vesicular acetylcholine transporter in the basal forebrain improves cognitive impairment in chronic cerebral hypoperfusion rats by modulating theta oscillations in the hippocampus.

The cholinergic transmission in the medial septum and ventral limb of the diagonal band of broca (MS/VDB)-hippocampal circuit and its associated theta oscillations play a crucial role in chronic cerebral hypoperfusion (CCH)-related cognitive impairment. However, the contribution and mechanism of the vesicular acetylcholine transporter (VAChT), a vital protein that regulates acetylcholine (ACh) release, in CCH-related cognitive impairment are not well understood. To investigate this, we established a rat model of CCH by performing 2-vessel occlusion (2-VO) and overexpressed VAChT in the MS/VDB via stereotaxic injection of adeno-associated virus (AAV). We evaluated the cognitive function of the rats using the Morris Water Maze (MWM) and Novel Object Recognition Test (NOR). We employed enzyme-linked immunosorbent assay (ELISA), Western blot (WB), and immunohistochemistry (IHC) to assess hippocampal cholinergic levels. We also conducted in vivo local field potentials (LFPs) recording experiments to evaluate changes in hippocampal theta oscillations and synchrony. Our findings showed that VAChT overexpression shortened the escape latency in the hidden platform test, increased swimming time in the platform quadrant in probe trains, and increased the recognition index (RI) in NOR. Moreover, VAChT overexpression increased hippocampal cholinergic levels, improved theta oscillations, and improved the synchrony of theta oscillations between CA1 and CA3 in CCH rats. These results suggest that VAChT plays a protective role in CCH-induced cognitive deficits by regulating cholinergic transmission in the MS/VDB-hippocampal circuit and promoting hippocampal theta oscillations. Therefore, VAChT could be a promising therapeutic target for treating CCH-related cognitive impairments.

Distribution of cholinergic nerve terminals in the aged human brain measured with [18F]FEOBV PET and its correlation with histological data.

INTRODUCTION: [18F]fluoroetoxybenzovesamicol ([18F]FEOBV) is a positron emission topography (PET) tracer for the vesicular acetylcholine transporter (VAChT), a protein located predominantly in synaptic vesicles in cholinergic nerve terminals. We aimed to use [18F]FEOBV PET to study the cholinergic topography of the healthy human brain. MATERIALS AND METHODS: [18F]FEOBV PET brain data volumes of healthy elderly humans were normalized to standard space and intensity-normalized to the white matter. Stereotactic atlases of regions of interest were superimposed to describe and quantify tracer distribution. The spatial distribution of [18F]FEOBV PET uptake was compared with histological and gene expression data. RESULTS: Twenty participants of both sexes and a mean age of 73.9 ± 6.0 years, age-range [64; 86], were recruited. Highest tracer binding was present in the striatum, some thalamic nuclei, and the basal forebrain. Intermediate binding was found in most nuclei of the brainstem, thalamus, and hypothalamus; the vermis and flocculonodular lobe; and the hippocampus, amygdala, insula, cingulate, olfactory cortex, and Heschl's gyrus. Lowest binding was present in most areas of the cerebral cortex, and in the cerebellar nuclei and hemispheres. The spatial distribution of tracer correlated with immunohistochemical post-mortem data, as well as with regional expression levels of SLC18A3, the VAChT coding gene. DISCUSSION: Our in vivo findings confirm the regional cholinergic distribution in specific brain structures as described post-mortem. A positive spatial correlation between tracer distribution and regional gene expression levels further corroborates [18F]FEOBV PET as a validated tool for in vivo cholinergic imaging. The study represents an advancement in the continued efforts to delineate the spatial topography of the human cholinergic system in vivo.

Maintenance of neurotransmitter identity by Hox proteins through a homeostatic mechanism.

Hox transcription factors play fundamental roles during early patterning, but they are also expressed continuously, from embryonic stages through adulthood, in the nervous system. However, the functional significance of their sustained expression remains unclear. In C. elegans motor neurons (MNs), we find that LIN-39 (Scr/Dfd/Hox4-5) is continuously required during post-embryonic life to maintain neurotransmitter identity, a core element of neuronal function. LIN-39 acts directly to co-regulate genes that define cholinergic identity (e.g., unc-17/VAChT, cho-1/ChT). We further show that LIN-39, MAB-5 (Antp/Hox6-8) and the transcription factor UNC-3 (Collier/Ebf) operate in a positive feedforward loop to ensure continuous and robust expression of cholinergic identity genes. Finally, we identify a two-component design principle for homeostatic control of Hox gene expression in adult MNs: Hox transcriptional autoregulation is counterbalanced by negative UNC-3 feedback. These findings uncover a noncanonical role for Hox proteins during post-embryonic life, critically broadening their functional repertoire from early patterning to the control of neurotransmitter identity.

Development and preclinical evaluation of [18F]FBVM as a new potent PET tracer for vesicular acetylcholine transporter.

Age-related neurodegenerative diseases have in common the occurrence of cognitive impairment, a highly incapacitating process that involves the cholinergic neurotransmission system. The vesicular acetylcholine transporter (VAChT) positron emission tomography (PET) tracer [18F]fluoroethoxybenzovesamicol ((-)-[18F]FEOBV) has recently demonstrated its high value to detect alterations of the cholinergic system in Alzheimer's disease, Parkinson's disease and dementia with Lewy body. We present here the development of the new vesamicol derivative tracer (-)-(R,R)-5-[18F]fluorobenzovesamicol ((-)[18F]FBVM) that we compared to (-)[18F]FEOBV in the same experimental conditions. We show that: i) in vitro affinity for the VAChT was 50-fold higher for (-)FBVM (Ki = 0.9 ± 0.3 nM) than for (-)FEOBV (Ki = 61 ± 2.8 nM); ii) in vivo in rats, a higher signal-to-noise specific brain uptake and a lower binding to plasma proteins and peripheral defluorination were obtained for (-)[18F]FBVM compared to (-)[18F]FEOBV. Our findings demonstrate that (-)[18F]FBVM is a highly promising PET imaging tracer which could be sufficiently sensitive to detect in humans the cholinergic denervation that occurs in brain areas having a low density of VAChT such as the cortex and hippocampus.

The Heart's Pacemaker Mimics Brain Cytoarchitecture and Function: Novel Interstitial Cells Expose Complexity of the SAN.

BACKGROUND: The sinoatrial node (SAN) of the heart produces rhythmic action potentials, generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of a hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)-expressing pacemaker cell meshwork, which merges with a network of connexin 43+/F-actin+ cells. It is also known that sympathetic and parasympathetic innervation create an autonomic plexus in the SAN that modulates heart rate and rhythm. However, the anatomical details of the interaction of this plexus with the pacemaker cell meshwork have yet to be described. OBJECTIVES: This study sought to describe the 3-dimensional cytoarchitecture of the mouse SAN, including autonomic innervation, peripheral glial cells, and pacemaker cells. METHODS: The cytoarchitecture of SAN whole-mount preparations was examined by three-dimensional confocal laser-scanning microscopy of triple immunolabeled with combinations of antibodies for HCN4, S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), choline acetyltransferase, or vesicular acetylcholine transporter, and tyrosine hydroxylase, and transmission electron microscopy. RESULTS: The SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells and a novel S100B+/GFAP- interstitial cell population, with a unique morphology and a distinct distribution pattern, creating complex interactions with other cell types in the node, particularly with HCN4-expressing cells. Transmission electron microscopy identified a similar population of interstitial cells as telocytes, which appeared to secrete vesicles toward pacemaker cells. Application of S100B to SAN preparations desynchronized Ca2+ signaling in HCN4-expressing cells and increased variability in SAN impulse rate and rhythm. CONCLUSIONS: The autonomic plexus, peripheral glial cell web, and a novel S100B+/GFAP- interstitial cell type embedded within the HCN4+ cell meshwork increase the structural and functional complexity of the SAN and provide a new regulatory pathway of rhythmogenesis.

The distribution and chemical coding of urinary bladder trigone-projecting neurons in testicular and aorticorenal ganglia in male pigs.

Combined retrograde tracing and double-labelling immunofluorescence were used to investigate the distribution and chemical coding of neurons in testicular (TG) and aorticoerenal (ARG) ganglia supplying the urinary bladder trigone (UBT) in juvenile male pigs (n=4, 12 kg. of body weight). Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the bladder trigone under pentobarbital anesthesia. After three weeks all the pigs were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde. TG and ARG, were collected and processed for double-labelling immunofluorescence. The expression of tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), nitric oxide synthase (NOS) and vesicular acetylcholine transporter (VAChT) were investigated. The cryostat sections were examined with a Zeiss LSM 710 confocal microscope equipped with adequate filter blocks. The TG and ARG were found to contain many FB-positive neurons projecting to the UBT (UBT-PN). The UBT-PN were distributed in both TG and ARG. The majority of them were found in the right ganglia, mostly in TG. Immunohistochemistry disclosed that the vast majority of UBT-PN were noradrenergic (TH- and/or DBH-positive). Many noradrenergic neurons contained also immunoreactivity to NPY, SOM or GAL. Most of the UBT-PN were supplied with VAChT-, or NOS- IR (immunoreactive) varicose nerve fibres. This study has revealed a relatively large population of differently coded prevertebral neurons projecting to the porcine urinary bladder. As judged from their neurochemical organization these nerve cells constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.

Carnosol Reduced Pathogenic Protein Aggregation and Cognitive Impairment in Neurodegenerative Diseases Models via Improving Proteostasis and Ameliorating Mitochondrial Disorders.

Neurodegenerative diseases (NDs) such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are incurable diseases with progressive loss of neural function and require urgent development of effective treatments. Carnosol (CL) reportedly has a pharmacological effect in the prevention of dementia. Nevertheless, the mechanisms of CL's neuroprotection are not entirely clear. The present study aimed to investigate the effects and mechanisms of CL-mediated neuroprotection through Caenorhabditis elegans models. First, CL restored ND protein homeostasis via inhibiting the IIS pathway, regulating MAPK signaling, and simultaneously activating molecular chaperone, thus inhibiting amyloid peptide (Aß), polyglutamine (polyQ), and α-synuclein (α-syn) deposition and reducing protein disruption-mediated behavioral and cognitive impairments as well as neuronal damages. Furthermore, CL could repair mitochondrial structural damage via improving the mitochondrial membrane protein function and mitochondrial structural homeostasis and improve mitochondrial functional defects via increasing adenosine triphosphate contents, mitochondrial membrane potential, and reactive oxygen species levels, suggesting that CL could improve the ubiquitous mitochondrial defects in NDs. More importantly, we found that CL activated mitochondrial kinetic homeostasis related genes to improve the mitochondrial homeostasis and dysfunction in NDs. Meanwhile, CL up-regulated unc-17, cho-1, and cha-1 genes to alleviate Aß-mediated cholinergic neurological disorders and activated Notch signaling and the Wnt pathway to diminish polyQ- and α-syn-induced ASH neurons as well as dopaminergic neuron damages. Overall, our study clarified the beneficial anti-ND neuroprotective effects of CL in different aspects and provided new insights into developing CL into products with preventive and therapeutic effects on NDs.

Long-term nucleus basalis cholinergic depletion induces attentional deficits and impacts cortical neurons and BDNF levels without affecting the NGF synthesis.

Basal forebrain cholinergic neurons (BFCNs) represent the main source of cholinergic innervation to the cortex and hippocampus and degenerate early in Alzheimer's disease (AD) progression. Phenotypic maintenance of BFCNs depends on levels of mature nerve growth factor (mNGF) and mature brain-derived neurotrophic factor (mBDNF), produced by target neurons and retrogradely transported to the cell body. Whether a reciprocal interaction where BFCN inputs impact neurotrophin availability and affect cortical neuronal markers remains unknown. To address our hypothesis, we immunolesioned the nucleus basalis (nb), a basal forebrain cholinergic nuclei projecting mainly to the cortex, by bilateral stereotaxic injection of 192-IgG-Saporin (the cytotoxin Saporin binds p75ntr receptors expressed exclusively by BFCNs) in 2.5-month-old Wistar rats. At 6 months post-lesion, Saporin-injected rats (SAP) showed an impairment in a modified version of the 5-Choice Serial Reaction Time Task (5-choice task). Postmortem analyses of the brain revealed a reduction of Choline Acetyltransferase-immunoreactive neurons compared to wild-type controls. A diminished number of cortical vesicular acetylcholine transporter-immunoreactive boutons was accompanied by a reduction in BDNF mRNA, mBDNF protein levels, markers of glutamatergic (vGluT1), and GABAergic (GAD65) neurons in the SAP-group compared to the controls. NGF mRNA, NGF precursor, and mNGF protein levels were not affected. Additionally, cholinergic markers correlated with the attentional deficit and BDNF levels. Our findings demonstrate that while cholinergic nb loss impairs cognition and reduces cortical neuron markers, it produces differential effects on neurotrophin availability, affecting BDNF but not NGF levels.

Cholinergic brain network deficits associated with vestibular sensory conflict deficits in Parkinson's disease: correlation with postural and gait deficits.

To examine regional cerebral vesicular acetylcholine transporter (VAChT) ligand [18F]fluoroethoxybenzovesamicol ([18F]-FEOBV) PET binding in Parkinson' disease (PD) patients with and without vestibular sensory conflict deficits (VSCD). To examine associations between VSCD-associated cholinergic brain deficits and postural instability and gait difficulties (PIGD). PD persons (M70/F22; mean age 67.6 ± 7.4 years) completed clinical assessments for imbalance, falls, freezing of gait (FoG), modified Romberg sensory conflict testing, and underwent VAChT PET. Volumes of interest (VOI)-based analyses included detailed thalamic and cerebellar parcellations. VSCD-associated VAChT VOI selection used stepwise logistic regression analysis. Vesicular monoamine transporter type 2 (VMAT2) [11C]dihydrotetrabenazine (DTBZ) PET imaging was available in 54 patients. Analyses of covariance were performed to compare VSCD-associated cholinergic deficits between patients with and without PIGD motor features while accounting for confounders. PET sampling passed acceptance criteria in 73 patients. This data-driven analysis identified cholinergic deficits in five brain VOIs associating with the presence of VSCD: medial geniculate nucleus (MGN) (P < 0.0001), para-hippocampal gyrus (P = 0.0043), inferior nucleus of the pulvinar (P = 0.047), fusiform gyrus (P = 0.035) and the amygdala (P = 0.019). Composite VSCD-associated [18F]FEOBV-binding deficits in these 5 regions were significantly lower in patients with imbalance (- 8.3%, F = 6.5, P = 0.015; total model: F = 5.1, P = 0.0008), falls (- 6.9%, F = 4.9, P = 0.03; total model F = 4.7, P = 0.0015), and FoG (- 14.2%, F = 9.0, P = 0.0043; total model F = 5.8, P = 0.0003), independent of age, duration of disease, gender and nigrostriatal dopaminergic losses. Post hoc analysis using MGN VAChT binding as the single cholinergic VOI demonstrated similar significant associations with imbalance, falls and FoG. VSCD-associated cholinergic network changes localize to distinct structures involved in multi-sensory, in particular vestibular, and multimodal cognitive and motor integration brain regions. Relative clinical effects of VSCD-associated cholinergic network deficits were largest for FoG followed by postural imbalance and falls. The MGN was the most significant region identified.

Improvement of Cognitive Function in Ovariectomized Rats by Human Neural Stem Cells Overexpressing Choline Acetyltransferase via Secretion of NGF and BDNF.

Menopause is associated with memory deficits attributed to reduced serum estrogen levels. We evaluated whether an increase in brain-derived neurotrophic factor (BDNF) and nerve-growth factor (NGF) levels, through transplantation of choline acetyltransferase (ChAT)-overexpressing neural stem cells (F3.ChAT), improved learning and memory in ovariectomized rats. PD13 mouse neuronal primary culture cells were treated with estradiol or co-cultured with F3.ChAT cells; choline transporter1 (CHT1), ChAT, and vesicular acetylcholine transporter (VAChT) expression was evaluated using real-time PCR. The relationship between estrogen receptors (ERs) and neurotrophin family members was analyzed using immunohistochemistry. After the transplantation of F3.ChAT cells into OVx rats, we evaluated the memory, ACh level, and the expression of ER, neurotrophin family proteins, and cholinergic system. Estradiol upregulated CHT1, ChAT, and VAChT expression in ER; they were co-localized with BDNF, NGF, and TrkB. Co-culture with F3.ChAT upregulated CHT1, ChAT, and VAChT by activating the neurotrophin signalling pathway. Transplantation of F3.ChAT cells in OVX animals increased the ACh level in the CSF and improved memory deficit. In addition, it increased the expression of ERs, neurotrophin signaling, and the cholinergic system in the brains of OVX animals. Therefore, the estradiol deficiency induced memory loss by the down-regulation of the neurotrophin family and F3.ChAT could ameliorate the cognitive impairment owing to the loss or reduction of estradiol.

No Dopamine Agonist Modulation of Brain [18F]FEOBV Binding in Parkinson's Disease.

The [18F]fluoroethoxybenzovesamicol ([18F]FEOBV) positron emission tomography (PET) ligand targets the vesicular acetylcholine transporter. Recent [18F]FEOBV PET rodent studies suggest that regional brain [18F]FEOBV binding may be modulated by dopamine D2-like receptor agents. We examined associations of regional brain [18F]FEOBV PET binding in Parkinson's disease (PD) subjects without versus with dopamine D2-like receptor agonist drug treatment. PD subjects (n = 108; 84 males, 24 females; mean age 68.0 ± 7.6 [SD] years), mean disease duration of 6.0 ± 4.0 years, and mean Movement Disorder Society-revised Unified PD Rating Scale III 35.5 ± 14.2 completed [18F]FEOBV brain PET imaging. Thirty-eight subjects were taking dopamine D2-like agonists. Vesicular monoamine transporter type 2 [11C]dihydrotetrabenazine (DTBZ) PET was available in a subset of 54 patients. Subjects on dopamine D2-like agonists were younger, had a longer duration of disease, and were taking a higher levodopa equivalent dose (LED) compared to subjects not taking dopamine agonists. A group comparison between subjects with versus without dopamine D2-like agonist use did not yield significant differences in cortical, striatal, thalamic, or cerebellar gray matter [18F]FEOBV binding. Confounder analysis using age, duration of disease, LED, and striatal [11C]DTBZ binding also failed to show significant regional [18F]FEOBV binding differences between these two groups. Chronic D2-like dopamine agonist use in PD subjects is not associated with significant alterations of regional brain [18F]FEOBV binding.

Functional dissociation of behavioral effects from acetylcholine and glutamate released from cholinergic striatal interneurons.

In the striatum, cholinergic interneurons (CINs) have the ability to release both acetylcholine and glutamate, due to the expression of the vesicular acetylcholine transporter (VAChT) and the vesicular glutamate transporter 3 (VGLUT3). However, the relationship these neurotransmitters have in the regulation of behavior is not fully understood. Here we used reward-based touchscreen tests in mice to assess the individual and combined contributions of acetylcholine/glutamate co-transmission in behavior. We found that reduced levels of the VAChT from CINs negatively impacted dopamine signalling in response to reward, and disrupted complex responses in a sequential chain of events. In contrast, diminished VGLUT3 levels had somewhat opposite effects. When mutant mice were treated with haloperidol in a cue-based task, the drug did not affect the performance of VAChT mutant mice, whereas VGLUT3 mutant mice were highly sensitive to haloperidol. In mice where both vesicular transporters were deleted from CINs, we observed altered reward-evoked dopaminergic signalling and behavioral deficits that resemble, but were worse, than those in mice with specific loss of VAChT alone. These results demonstrate that the ability to secrete two different neurotransmitters allows CINs to exert complex modulation of a wide range of behaviors.

Change in the neurochemical signature and morphological development of the parvocellular isthmic projection to the avian tectum.

Neurons can change their classical neurotransmitters during ontogeny, sometimes going through stages of dual release. Here, we explored the development of the neurotransmitter identity of neurons of the avian nucleus isthmi parvocellularis (Ipc), whose axon terminals are retinotopically arranged in the optic tectum (TeO) and exert a focal gating effect upon the ascending transmission of retinal inputs. Although cholinergic and glutamatergic markers are both found in Ipc neurons and terminals of adult pigeons and chicks, the mRNA expression of the vesicular acetylcholine transporter, VAChT, is weak or absent. To explore how the Ipc neurotransmitter identity is established during ontogeny, we analyzed the expression of mRNAs coding for cholinergic (ChAT, VAChT, and CHT) and glutamatergic (VGluT2 and VGluT3) markers in chick embryos at different developmental stages. We found that between E12 and E18, Ipc neurons expressed all cholinergic mRNAs and also VGluT2 mRNA; however, from E16 through posthatch stages, VAChT mRNA expression was specifically diminished. Our ex vivo deposits of tracer crystals and intracellular filling experiments revealed that Ipc axons exhibit a mature paintbrush morphology late in development, experiencing marked morphological transformations during the period of presumptive dual vesicular transmitter release. Additionally, although ChAT protein immunoassays increasingly label the growing Ipc axon, this labeling was consistently restricted to sparse portions of the terminal branches. Combined, these results suggest that the synthesis of glutamate and acetylcholine, and their vesicular release, is complexly linked to the developmental processes of branching, growing and remodeling of these unique axons.

Homeostatic plasticity induced by increased acetylcholine release at the mouse neuromuscular junction.

At the neuromuscular junction (NMJ), changes to the size of the postsynaptic potential induce homeostatic compensation. At the Drosophila NMJ, increased glutamate release causes a compensatory decrease in quantal content, but it is unknown if this mechanism operates at the cholinergic mammalian NMJ. We addressed this question by recording endplate potentials (EPP) and muscle contraction in 3-month and 24-month ChAT-ChR2-EYFP mice that overexpress vesicular acetylcholine transporter and release more acetylcholine per vesicle. At 3 months, the quantal content of EPPs from ChAT-ChR2-EYFP mice were not different from WT controls, however tetanic depression was greater, and quantal size during high-frequency stimulation and the size of the readily releasable pool (RRP) were decreased. At 24 months of age, quantal content was reduced in ChAT-ChR2-EYFP mice, which normalized synaptic depression despite smaller RRP. The effect of pancuronium on indirect evoked muscle twitch was not different between groups. These results indicate that an increase in the amount of acetylcholine per vesicle induces two distinct age-dependent homeostatic mechanisms compensating excessive acetylcholine release.

Evaluation of radiolabeled acetylcholine synthesis and release in rat striatum.

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [3 H]choline and subsequent synthesis and release of [3 H]ACh were investigated in rat striatal segments. Incubation with [3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [3 H]choline and [3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [3 H]choline led predominantly to the accumulation of [3 H]ACh. Electrical stimulation and KCl depolarization selectively released [3 H]ACh but not [3 H]choline. [3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [3 H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca2+ dependent and exhibited slightly different sensitivities to N- and P-type Ca2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.

Brain Factor-7® Improves Cognitive Impairment Following Transient Ischemia and Reperfusion Injury in Gerbil Forebrain through Promoting Remyelination and Restoring Cholinergic and Glutamatergic Neurotransmission in the Hippocampus.

BACKGROUND: Ischemia and reperfusion injury in the brain triggers cognitive impairment which are accompanied by neuronal death, loss of myelin sheath and decline in neurotransmission. In this study, we investigated whether therapeutic administration of Brain Factor-7® (BF-7®; a silk peptide) in ischemic gerbils which were developed by transient (five minutes) ischemia and reperfusion in the forebrain (tFI/R) improved cognitive impairment. METHODS: Short-term memory and spatial memory functions were assessed by passive avoidance test and Barnes maze test, respectively. To examine neuronal change in the hippocampus, cresyl violet staining, immunohistochemistry for neuronal nuclei and fluoro Jade B histofluorescence were performed. We carried out immunohistochemistry for myelin basic protein (a marker for myelin) and receptor interacting protein (a marker for oligodendrocytes). Furthermore, immunohistochemistry for vesicular acetylcholine transporter (as a cholinergic transporter) and vesicular glutamate transporter 1 (as a glutamatergic synapse) was done. RESULTS: Administration of BF-7® significantly improved tFI/R-induced cognitive impairment. tFI/R-induced neuronal death was found in the Cornu Ammonis 1 (CA1) subfield of the hippocampus from five days after tFI/R. Treatment with BF-7® following tFI/R did not restore the death (loss) of CA1 neurons following tFI/R. However, BF-7® treatment to the ischemic gerbils significantly improved remyelination and proliferation of oligodendrocytes in the hippocampus with ischemic injury. Treatment with BF-7® to the ischemic gerbils significantly restored vesicular acetylcholine transporter-immunoreactive and vesicular glutamate transporter 1-immunoreactive structures in the hippocampus with ischemic injury. CONCLUSIONS: Based on these results, we suggest that BF-7® can be utilized for improving cognitive impairments induced by ischemic injury as an additive for health/functional foods and/or medicines.

Reduction of acetylcholine in the hippocampus of hippocampal cholinergic neurostimulating peptide precursor protein knockout mice.

The cholinergic efferent network from the medial septal nucleus to the hippocampus plays an important role in learning and memory processes. This cholinergic projection can generate theta oscillations in the hippocampus to encode novel information. Hippocampal cholinergic neurostimulating peptide (HCNP), which induces acetylcholine (Ach) synthesis in the medial septal nuclei of an explant culture system, was purified from the soluble fraction of postnatal rat hippocampus. HCNP is processed from the N-terminal region of a 186-amino acid, 21-kDa HCNP precursor protein, also known as Raf kinase inhibitory protein and phosphatidylethanolamine-binding protein 1. Here, we confirmed direct reduction of Ach release in the hippocampus of freely moving HCNP-pp knockout mice under an arousal state by the microdialysis method. The levels of vesicular acetylcholine transporter were also decreased in the hippocampus of these mice in comparison with those in control mice, suggesting there was decreased incorporation of Ach into the synaptic vesicle. These results potently indicate that HCNP may be a cholinergic regulator in the septo-hippocampal network.

Markers of the sympathetic, parasympathetic and sensory nervous system are altered in the human diabetic choroid.

BACKGROUND: We sought to evaluate alterations in markers of the autonomic nervous system in human diabetic choroid. METHODS: Eighteen eyeballs from subjects with diabetes and 22 eyeballs from subjects without diabetes were evaluated in this study. Synaptophysin, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DßH), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), vesicular monoamine transporter II (VMAT-2), vesicular acetylcholine transporter (VAChT), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) levels were detected by western blot analysis and immunofluorescence was performed in some cases. Furthermore, differences in adrenergic (α1- and ß2-subtypes) and cholinergic (M1 and M3) receptor levels between diabetic subjects and controls were noted. RESULTS: Decreased synaptophysin levels were found in diabetic choroids by western blot analysis and a reduction of synaptophysin-immunoreactive nerves was also found by immunofluorescence. Furthermore, a decrease of the levels of the key enzyme (TH) and transporter (VMAT2) of norepinephrine was evident both by western blot analysis and immunofluorescence. Additionally, increased NPY, VAChT, nNOS, and CGRP levels were observed in diabetic choroids. The levels of adrenergic (ß2 subtype) and acetylcholine (M1 subtype) receptors decreased in diabetic choroids, as shown by western blotting and although the differences in α1 and M3 were not significant, there was a downward trend. CONCLUSIONS: In the diabetic choroid, the levels of neurotransmitters, enzymes, and receptors associated with choroidal blood flow regulation are altered. These changes may affect the regulation of choroidal blood flow and may be associated with impaired retinal function and retinal pathology.