Topography of Cholinergic Nerve Terminal Vulnerability and Balance Self-Efficacy in Parkinson's Disease.

BACKGROUND: Postural instability and gait disturbances (PIGD) represent a significant cause of disability in Parkinson's disease (PD). Cholinergic system dysfunction has been implicated in falls in PD. The occurrence of falls typically results in fear of falling (FoF) that in turn may lead to poorer balance self-efficacy. Balance self-efficacy refers to one's level of confidence in their ability to balance while completing activities of daily living like getting dressed, bathing, and walking. Lower self-efficacy, or greater FoF during these activities is a function of motor, cognitive, and emotional impairments and may impact quality of life in PD. Unlike known cholinergic reduction, especially in the right lateral geniculate and caudate nuclei, little is known about the role of cholinergic transporters in FoF or mobility self-efficacy in PD. METHODS: [18F]fluoroethoxybenzovesamicol ([18F]FEOBV) positron emission tomography (PET) studies were conducted to assess vesicular acetylcholine transporter (VAChT) expression in 126 patients with PD (male (m) = 95, female (f) = 31). Participants had a mean age of 67.3 years (standard deviation (SD) = 7.1) and median Hoehn Yahr stage of 2.5. Patients also completed the Short Falls Efficacy Scale (sFES-I) as a survey measure of concerns about falling. [18F]FEOBV data were processed in Statistical Parametric Mapping (SPM) using a voxel-wise regression model with sFES-I scores as the outcome measure. RESULTS: Reduced [18F]FEOBV binding in tectum, metathalamic (lateral more than medial geniculate nuclei), thalamus proper, bilateral mesiotemporal (hippocampal, parahippocampal, fusiform gyri and fimbriae), and right cerebellar lobule VI significantly associated with higher sFES-I scores (p < 0.05, family-wise error (FWE) correction after Threshold-Free Cluster Enhancement (TFCE)). CONCLUSIONS: Unlike the more limited involvement of the brainstem-thalamic complex and caudate nuclei cholinergic topography associated with falls in PD, cholinergic reductions in the extended connectivity between the thalamic complex and the temporal limbic system via the fimbriae associates with FoF. Additional cholinergic changes were seen in the cerebellum. The temporal limbic system plays a role not only in episodic memory but also in spatial navigation, scene and contextual (e.g., emotional) processing. Findings may augur novel therapeutic approaches to treat poor mobility self-efficacy in PD. CLINICAL TRIAL REGISTRATION: No: NCT02458430. Registered 18 March, 2015, https://www. CLINICALTRIALS: gov/study/NCT02458430; No: NCT05459753. Registered 01 July, 2022, https://www. CLINICALTRIALS: gov/study/NCT05459753.

Urinary dysfunction after spinal cord injury: Comparing outcomes after thoracic spinal transection and contusion in the rat.

Spinal cord injury (SCI) above the lumbosacral spinal cord induces loss of voluntary control over micturition. Spinal cord transection (SCT) was the gold standard method to reproduce SCI in rodents, but its translational value is arguable and other experimental SCI methods need to be better investigated, including spinal cord contusion (SCC). At present, it is not fully investigated if urinary impairments arising after transection and contusion are comparable. To explore this, we studied bladder-reflex activity and lower urinary tract (LUT) and spinal cord innervation after SCT and different severities of SCC. Severe-contusion animals presented a longer spinal shock period and the tendency for higher residual volumes, followed by SCT and mild-contusion animals. Urodynamics showed that SCT animals presented higher basal and peak bladder pressures. Immunostaining against growth-associated protein-43 (GAP43) and calcitonin gene-related peptide (CGRP) at the lumbosacral spinal cord demonstrated that afferent sprouting is dependent on the injury model, reflecting the severity of the lesion, with a higher expression in SCT animals. In LUT organs, the expression of GAP43, CGRP cholinergic (vesicular acetylcholine transporter (VAChT)) and noradrenergic (tyrosine hydroxylase (TH)) markers was reduced after SCI in the LUT and lumbosacral cord, but only the lumbosacral expression of VAChT was dependent on the injury model. Overall, our findings demonstrate that changes in LUT innervation and function after contusion and transection are similar but result from distinct neuroplastic processes at the lumbosacral spinal cord. This may impact the development of new therapeutic options for urinary impairment arising after spinal cord insult.

Influence of ß2-adrenergic selective agonist formoterol on the motor unit of a mouse model of a congenital myasthenic syndrome with complete VAChT deletion.

Congenital Myasthenic Syndromes (CMS) are a set of genetic diseases that affect the neuromuscular transmission causing muscular weakness. The standard pharmacological treatment aims at ameliorating the myasthenic symptom by acetylcholinesterase inhibitors. Most patients respond well in the short and medium term, however, over time the beneficial effects rapidly fade, and the efficacy of the treatment diminishes. Increasing evidence shows that ß2-adrenergic agonists can be a suitable choice for the treatment of neuromuscular disorders, including CMS, as they promote beneficial effects in the neuromuscular system. The exact mechanism on which they rely is not completely understood, although patients and animal models respond well to the treatment, especially over extended periods. Here, we report the use of the long-lasting specific ß2-adrenergic agonist formoterol in a myasthenic mouse model (mnVAChT-KD), featuring deletion of VAChT (Vesicular Acetylcholine Transporter) specifically in the α-motoneurons. Our findings demonstrate that formoterol treatment (300 µg/kg/day; sc) for 30 days increased the neuromuscular junction area, induced skeletal muscle hypertrophy and altered fibre type composition in myasthenic mice. Interestingly, ß2-adrenergic agonists have shown efficacy even in the absence of ACh (acetylcholine). Our data provide important evidence supporting the potential of ß2-adrenergic agonists in treating neuromuscular disorders of pre-synaptic origin and characterized by disruptions in nerve-muscle communication, through a direct and beneficial action within the motor unit.

Comparison of the Influence of Bisphenol A and Bisphenol S on the Enteric Nervous System of the Mouse Jejunum.

Bisphenols are dangerous endocrine disruptors that pollute the environment. Due to their chemical properties, they are globally used to produce plastics. Structural similarities to oestrogen allow bisphenols to bind to oestrogen receptors and affect internal body systems. Most commonly used in the plastic industry is bisphenol A (BPA), which also has negative effects on the nervous, immune, endocrine, and cardiovascular systems. A popular analogue of BPA-bisphenol S (BPS) also seems to have harmful effects similar to BPA on living organisms. Therefore, with the use of double immunofluorescence labelling, this study aimed to compare the effect of BPA and BPS on the enteric nervous system (ENS) in mouse jejunum. The study showed that both studied toxins impact the number of nerve cells immunoreactive to substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), the neuronal isoform of nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VAChT). The observed changes were similar in the case of both tested bisphenols. However, the influence of BPA showed stronger changes in neurochemical coding. The results also showed that long-term exposure to BPS significantly affects the ENS.

Oral Exposure to Microplastics Affects the Neurochemical Plasticity of Reactive Neurons in the Porcine Jejunum.

Plastics are present in almost every aspect of our lives. Polyethylene terephthalate (PET) is commonly used in the food industry. Microparticles can contaminate food and drinks, posing a threat to consumers. The presented study aims to determine the effect of microparticles of PET on the population of neurons positive for selected neurotransmitters in the enteric nervous system of the jejunum and histological structure. An amount of 15 pigs were divided into three groups (control, receiving 0.1 g, and 1 g/day/animal orally). After 28 days, fragments of the jejunum were collected for immunofluorescence and histological examination. The obtained results show that histological changes (injury of the apical parts of the villi, accumulations of cellular debris and mucus, eosinophil infiltration, and hyperaemia) were more pronounced in pigs receiving a higher dose of microparticles. The effect on neuronal nitric oxide synthase-, and substance P-positive neurons, depends on the examined plexus and the dose of microparticles. An increase in the percentage of galanin-positive neurons and a decrease in cocaine and amphetamine-regulated transcript-, vesicular acetylcholine transporter-, and vasoactive intestinal peptide-positive neurons do not show such relationships. The present study shows that microparticles can potentially have neurotoxic and pro-inflammatory effects, but there is a need for further research to determine the mechanism of this process and possible further effects.

The clinicopathological and prognostic significance of autonomic nerves in salivary duct carcinoma.

Many researchers have focused on the role of the autonomic nervous system in the tumor microenvironment. Autonomic nerves include the sympathetic and parasympathetic nerves, which are known to induce cancer growth and metastasis. However, in salivary duct carcinoma (SDC), a rare and highly malignant tumor, the issue should be investigated from both biological and therapeutic perspectives. We explored the clinicopathological and prognostic implications of the autonomic nerves in 129 SDCs. Immunohistochemistry was performed to determine the nature of each nerve using antibodies against S100, tyrosine hydroxylase (TH) as a sympathetic marker, and vesicular acetylcholine transporter (VAChT) as a parasympathetic marker. The area of each marker-positive nerve was digitized and evaluated quantitatively. Double immunofluorescence for TH and VAChT was performed in selected cases. The expression of the secreted neurotrophins was also examined. S100-positive nerves were present in the cancer tissue in 94 of 129 cases (72.9%). Among them, TH-positive sympathetic nerves and/or VAChT-positive parasympathetic nerves were identified in 92 cases (97.9%), and 59 cases (62.8%) had TH/VAChT-co-expressing nerves. Double immunofluorescence revealed a mosaic pattern of sympathetic and parasympathetic fibers in co-expressing nerve bundles. The presence of autonomic nerves, regardless of their area, was significantly associated with aggressive histological features, advanced T/N classification, and a poor prognosis, with shorter disease-free and overall survival. There was an association between some tumor immune microenvironment-related markers and the autonomic nerve status, but not the latter and the secreted neurotrophin expression. This study suggests that autonomic nerves might play a role in the progression of SDC.

PET Quantification of [18F]VAT in Human Brain and Its Test-Retest Reproducibility and Age Dependence.

Molecular imaging of brain vesicular acetylcholine transporter provides a biomarker to explore cholinergic systems in humans. We aimed to characterize the distribution of, and optimize methods to quantify, the vesicular acetylcholine transporter-specific tracer (-)-(1-(8-(2-[18F]fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-piperidin-4-yl)(4-fluorophenyl)methanone ([18F]VAT) in the brain using PET. Methods: Fifty-two healthy participants aged 21-97 y had brain PET with [18F]VAT. [3H]VAT autoradiography identified brain areas devoid of specific binding in cortical white matter. PET image-based white matter reference region size, model start time, and duration were optimized for calculations of Logan nondisplaceable binding potential (BPND). Ten participants had 2 scans to determine test-retest variability. Finally, we analyzed age-dependent differences in participants. Results: [18F]VAT was widely distributed in the brain, with high striatal, thalamic, amygdala, hippocampal, cerebellar vermis, and regionally specific uptake in the cerebral cortex. [3H]VAT autoradiography-specific binding and PET [18F]VAT uptake were low in white matter. [18F]VAT SUVs in the white matter reference region correlated with age, requiring stringent erosion parameters. Logan BPND estimates stabilized using at least 40 min of data starting 25 min after injection. Test-retest variability had excellent reproducibility and reliability in repeat BPND calculations for 10 participants (putamen, 6.8%; r > 0.93). We observed age-dependent decreases in the caudate and putamen (multiple comparisons corrected) and in numerous cortical regions. Finally, we provide power tables to indicate potential mean differences that can be detected between 2 groups of participants. Conclusion: These results validate a reference region for BPND calculations and demonstrate the viability, reproducibility, and utility of using the [18F]VAT tracer in humans to quantify cholinergic pathways.

Imaging the Vesicular Acetylcholine Transporter in Schizophrenia: A Positron Emission Tomography Study Using [18F]-VAT.

BACKGROUND: Despite longstanding interest in the central cholinergic system in schizophrenia (SCZ), cholinergic imaging studies with patients have been limited to receptors. Here, we conducted a proof-of-concept positron emission tomography study using [18F]-VAT, a new radiotracer that targets the vesicular acetylcholine transporter as a proxy measure of acetylcholine transmission capacity, in patients with SCZ and explored relationships of vesicular acetylcholine transporter with clinical symptoms and cognition. METHODS: A total of 18 adult patients with SCZ or schizoaffective disorder (the SCZ group) and 14 healthy control participants underwent a positron emission tomography scan with [18F]-VAT. Distribution volume (VT) for [18F]-VAT was derived for each region of interest, and group differences in VT were assessed with 2-sample t tests. Functional significance was explored through correlations between VT and scores on the Positive and Negative Syndrome Scale and a computerized neurocognitive battery (PennCNB). RESULTS: No group differences in [18F]-VAT VT were observed. However, within the SCZ group, psychosis symptom severity was positively associated with VT in multiple regions of interest, with the strongest effects in the hippocampus, thalamus, midbrain, cerebellum, and cortex. In addition, in the SCZ group, working memory performance was negatively associated with VT in the substantia innominata and several cortical regions of interest including the dorsolateral prefrontal cortex. CONCLUSIONS: In this initial study, the severity of 2 important features of SCZ-psychosis and working memory deficit-was strongly associated with [18F]-VAT VT in several cortical and subcortical regions. These correlations provide preliminary evidence of cholinergic activity involvement in SCZ and, if replicated in larger samples, could lead to a more complete mechanistic understanding of psychosis and cognitive deficits in SCZ and the development of therapeutic targets.

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.

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.

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.

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.

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.