Retrograde Axonal Transport of Neurotrophins in Basal Forebrain Cholinergic Neurons.

Axonal transport is key for the survival and function of all neurons. This process is especially important in basal forebrain cholinergic neurons due to their extremely long and diffuse axonal projections. These neurons are critical for learning and memory and degenerate rapidly in age-related neurodegenerative disorders like Alzheimer's and Parkinson's disease. The vulnerability of these neurons to age-related neurodegeneration may be partially attributed to their reliance on retrograde axonal transport for neurotrophic support. Unfortunately, little is known about the molecular biology underlying the retrograde transport dynamics of these neurons due to the difficulty associated with their maintenance in vitro. Here, we outline a protocol for culturing primary rodent basal forebrain cholinergic neurons in microfluidic chambers, devices designed specifically for the study of axonal transport in vitro. We outline protocols for labeling neurotrophins and tracking neurotrophin transport in these neurons. Our protocols can also be used to study axonal transport in other types of primary neurons such as cortical and hippocampal neurons.

circRNA Acbd6 promotes neural stem cell differentiation into cholinergic neurons via the miR-320-5p-Osbpl2 axis.

Neural stem cells (NSCs) persist in the dentate gyrus of the hippocampus into adulthood and are essential for both neurogenesis and neural circuit integration. Exosomes have also been shown to play vital roles in regulating biological processes of receptor cells as a medium for cell-to-cell communication signaling molecules. The precise molecular mechanisms of exosome-mediated signaling, however, remain largely unknown. Here, we found that exosomes produced by denervated hippocampi following fimbria-fornix transection could promote the differentiation of hippocampal neural precursor cells into cholinergic neurons in coculture with NSCs. Furthermore, we found that 14 circular RNAs (circRNAs) were upregulated in hippocampal exosomes after fimbria-fornix transection using high-throughput RNA-Seq technology. We further characterized the function and mechanism by which the upregulated circRNA Acbd6 (acyl-CoA-binding domain-containing 6) promoted the differentiation of NSCs into cholinergic neurons using RT-quantitative PCR, Western blot, ELISA, flow cytometry, immunohistochemistry, and immunofluorescence assay. By luciferase reporter assay, we demonstrated that circAcbd6 functioned as an endogenous miR-320-5p sponge to inhibit miR-320-5p activity, resulting in increased oxysterol-binding protein-related protein 2 expression with subsequent facilitation of NSC differentiation. Taken together, our results suggest that circAcbd6 promotes differentiation of NSCs into cholinergic neurons via miR-320-5p/oxysterol-binding protein-related protein 2 axis, which contribute important insights to our understanding of how circRNAs regulate neurogenesis.

(-)-Epigallocatechin-3-Gallate Diminishes Intra-and Extracellular Amyloid-Induced Cytotoxic Effects on Cholinergic-like Neurons from Familial Alzheimer's Disease PSEN1 E280A.

Alzheimer's disease (AD) is a complex neurodegenerative disease characterized by functional disruption, death of cholinergic neurons (ChNs) because of intracellular and extracellular Aß aggregates, and hyperphosphorylation of protein TAU (p-TAU). To date, there are no efficient therapies against AD. Therefore, new therapies for its treatment are in need. The goal of this investigation was to evaluate the effect of the polyphenol epigallocatechin-3-gallate (EGCG) on cholinergic-like neurons (ChLNs) bearing the mutation E280A in PRESENILIN 1 (PSEN1 E280A). To this aim, wild-type (WT) and PSEN1 E280A ChLNs were exposed to EGCG (5-50 µM) for 4 days. Untreated or treated neurons were assessed for biochemical and functional analysis. We found that EGCG (50 µM) significantly inhibited the aggregation of (i)sAPPßf, blocked p-TAU, increased ∆Ψm, decreased oxidation of DJ-1 at residue Cys106-SH, and inhibited the activation of transcription factor c-JUN and P53, PUMA, and CASPASE-3 in mutant ChLNs compared to WT. Although EGCG did not reduce (e)Aß42, the polyphenol reversed Ca2+ influx dysregulation as a response to acetylcholine (ACh) stimuli in PSEN1 E280A ChLNs, inhibited the activation of transcription factor NF-κB, and reduced the secretion of pro-inflammatory IL-6 in wild-type astrocyte-like cells (ALCs) when exposed to mutant ChLNs culture supernatant. Taken together, our findings suggest that the EGCG might be a promising therapeutic approach for the treatment of FAD.

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons.

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Regulation of coordinated muscular relaxation in Drosophila larvae by a pattern-regulating intersegmental circuit.

Typical patterned movements in animals are achieved through combinations of contraction and delayed relaxation of groups of muscles. However, how intersegmentally coordinated patterns of muscular relaxation are regulated by the neural circuits remains poorly understood. Here, we identify Canon, a class of higher-order premotor interneurons, that regulates muscular relaxation during backward locomotion of Drosophila larvae. Canon neurons are cholinergic interneurons present in each abdominal neuromere and show wave-like activity during fictive backward locomotion. Optogenetic activation of Canon neurons induces relaxation of body wall muscles, whereas inhibition of these neurons disrupts timely muscle relaxation. Canon neurons provide excitatory outputs to inhibitory premotor interneurons. Canon neurons also connect with each other to form an intersegmental circuit and regulate their own wave-like activities. Thus, our results demonstrate how coordinated muscle relaxation can be realized by an intersegmental circuit that regulates its own patterned activity and sequentially terminates motor activities along the anterior-posterior axis.

The influence of castration on intramural neurons of the urinary bladder trigone in male pigs.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the intramural ganglia of the urinary bladder trigone (UBT) in male pigs using double-labeling immunohistochemistry. The ganglia were examined in intact (IP) 7-day-old (castration day) pigs, and at 3 and 6 months after surgery. In IP and control (3- and 6-month-old noncastrated pigs) groups, virtually, all neurons were adrenergic (68%) or cholinergic (32%) in nature. Many of them (32%, 51%, and 81%, respectively; 56%, 75%, and 85% adrenergic; and 32%, 52%, and 65% cholinergic, respectively) stained for the androgen receptor (AR), and only a small number of nerve cells were caspase-3 (CASP-3)-positive. In 3- and 6-month-old castrated pigs, an excessive loss (87.6% and 87.5%, respectively) of neurons and intraganglionic nerve fibers was observed. The majority of the surviving adrenergic (61% and 72%, respectively) and many cholinergic (41% and 31%, respectively) neurons expressed CASP-3 and were also AR-positive (61% and 66%, and 40% and 36%, respectively). This study revealed for the first time the excessive loss of intramural UBT neurons following castration, which could have resulted from apoptosis induced by androgen deprivation.

Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord.

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we develop a targeted single nuclear RNA sequencing approach and use it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their rich diversity. Specifically, visceral motor neurons, which provide autonomic control, can be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons is also reflected in our analysis with alpha, gamma, and a third subtype, possibly corresponding to the elusive beta motor neurons, clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.

Distinct proportions of cholinergic neurons in the rat prepositus hypoglossi nucleus according to their cerebellar projection targets.

Cerebellar functions are modulated by cholinergic inputs, the density of which varies among cerebellar regions. Although the prepositus hypoglossi nucleus (PHN), a brainstem structure involved in controlling gaze holding, is known as one of the major sources of these cholinergic inputs, the proportions of cholinergic neurons in PHN projections to distinct cerebellar regions have not been quantitatively analyzed. In this study, we identified PHN neurons projecting to the cerebellum by applying retrograde labeling with dextran-conjugated Alexa 488 in choline acetyltransferase (ChAT)-tdTomato transgenic rats and compared the proportion of cholinergic PHN neurons in the PHN projections to four different regions of the cerebellum, namely the flocculus (FL), the uvula and nodulus (UN), lobules III-V in the vermis (VM), and the hemispheric paramedian lobule and crus 2 (PC). In the PHN, the percentage of cholinergic PHN neurons was lower in the projection to the FL than in the projection to the UN, VM or PC. Preposito-cerebellar neurons, except for preposito-FL neurons, included different proportions of cholinergic neurons at different rostrocaudal positions in the PHN. These results suggest that cholinergic PHN neurons project to not only the vestibulocerebellum but also the anterior vermis and posterior hemisphere and that the proportion of cholinergic neurons among projection neurons from the PHN differs depending on cerebellar target areas and the rostro-caudal regions of the PHN. This study provides insights regarding the involvement of cerebellar cholinergic networks in gaze holding.

Distribution of the cholinergic nuclei in the brain of the weakly electric fish, Apteronotus leptorhynchus: Implications for sensory processing.

Acetylcholine acts as a neurotransmitter/neuromodulator of many central nervous system processes such as learning and memory, attention, motor control, and sensory processing. The present study describes the spatial distribution of cholinergic neurons throughout the brain of the weakly electric fish, Apteronotus leptorhynchus, using in situ hybridization of choline acetyltransferase mRNA. Distinct groups of cholinergic cells were observed in the telencephalon, diencephalon, mesencephalon, and hindbrain. These included cholinergic cell groups typically identified in other vertebrate brains, for example, motor neurons. Using both in vitro and ex vivo neuronal tracing methods, we identified two new cholinergic connections leading to novel hypotheses on their functional significance. Projections to the nucleus praeeminentialis (nP) arise from isthmic nuclei, possibly including the nucleus lateralis valvulae (nLV) and the isthmic nucleus (nI). The nP is a central component of all electrosensory feedback pathways to the electrosensory lateral line lobe (ELL). We have previously shown that some neurons in nP, TS, and tectum express muscarinic receptors. We hypothesize that, based on nLV/nI cell responses in other teleosts and isthmic connectivity in A. leptorhynchus, the isthmic connections to nP, TS, and tectum modulate responses to electrosensory and/or visual motion and, in particular, to looming/receding stimuli. In addition, we found that the octavolateral efferent (OE) nucleus is the likely source of cholinergic fibers innervating the ELL. In other teleosts, OE inhibits octavolateral hair cells during locomotion. In gymnotiform fish, OE may also act on the first central processing stage and, we hypothesize, implement corollary discharge modulation of electrosensory processing during locomotion.

A Simple Differentiation Protocol for Generation of Induced Pluripotent Stem Cell-Derived Basal Forebrain-Like Cholinergic Neurons for Alzheimer's Disease and Frontotemporal Dementia Disease Modeling.

The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.

Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and ß-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

Neuroprotective Role of Dietary Supplementation with Omega-3 Fatty Acids in the Presence of Basal Forebrain Cholinergic Neurons Degeneration in Aged Mice.

As major components of neuronal membranes, omega-3 polyunsaturated fatty acids (n-3 PUFA) exhibit a wide range of regulatory functions. Recent human and animal studies indicate that n-3 PUFA may exert beneficial effects on aging processes. Here we analyzed the neuroprotective influence of n-3 PUFA supplementation on behavioral deficits, hippocampal neurogenesis, volume loss, and astrogliosis in aged mice that underwent a selective depletion of basal forebrain cholinergic neurons. Such a lesion represents a valid model to mimic a key component of the cognitive deficits associated with dementia. Aged mice were supplemented with n-3 PUFA or olive oil (as isocaloric control) for 8 weeks and then cholinergically depleted with mu-p75-saporin immunotoxin. Two weeks after lesioning, mice were behaviorally tested to assess anxious, motivational, social, mnesic, and depressive-like behaviors. Subsequently, morphological and biochemical analyses were performed. In lesioned aged mice the n-3 PUFA pre-treatment preserved explorative skills and associative retention memory, enhanced neurogenesis in the dentate gyrus, and reduced volume and VAChT levels loss as well as astrogliosis in hippocampus. The present findings demonstrating that n-3 PUFA supplementation before cholinergic depletion can counteract behavioral deficits and hippocampal neurodegeneration in aged mice advance a low-cost, non-invasive preventive tool to enhance life quality during aging.

Characterization of three-dimensional rat central nervous system culture maturation, with applications to monitor cholinergic integrity.

Studying age-related neuropathologies in vitro requires a three-dimensional (3D) culture system presenting mature phenotypes. In this study, we aimed to determine whether aged reaggregate cultures physiologically represent mature brain tissue. Results support that embryo-derived rat central nervous system (CNS) reaggregate cultures develop into mature-like tissues, comparable to in vivo maturation, including the following characteristics: (a) progressive reduction in cell proliferation (reduced anti-Ki-67 immunoreactivity), (b) progressive restriction of long neurite growth potential (as explant cultures), and (c) increased and sustained synaptic enzyme (acetylcholine esterase, AChE) activity. The acquisition of mature-like reaggregate cultures has allowed us to pursue the hypothesis that the physiological integrity of 3D CNS cultures may be monitored by synaptic enzyme activity. To assess this hypothesis, mature-like reaggregates were exposed to H2 O2 , glutamate, or amyloid ß(1-42); each resulted in diminished AChE activity. H2 O2 exposure resulted in nuclear fragmentation. Glutamate and amyloid ß(1-42) exposure resulted in acetylcholine content reduction. Simultaneous reduction of AChE activity and acetylcholine content verified diminished cholinergic integrity. This scheme exploiting synapse enzyme activity of mature-like 3D CNS tissue is therefore applicable to age-related neuropathology research including in vitro screening of conditions potentially affecting synapse integrity, including the promotion of dementia.

A terminal selector prevents a Hox transcriptional switch to safeguard motor neuron identity throughout life.

To become and remain functional, individual neuron types must select during development and maintain throughout life their distinct terminal identity features, such as expression of specific neurotransmitter receptors, ion channels and neuropeptides. Here, we report a molecular mechanism that enables cholinergic motor neurons (MNs) in the C. elegans ventral nerve cord to select and maintain their unique terminal identity. This mechanism relies on the dual function of the conserved terminal selector UNC-3 (Collier/Ebf). UNC-3 synergizes with LIN-39 (Scr/Dfd/Hox4-5) to directly co-activate multiple terminal identity traits specific to cholinergic MNs, but also antagonizes LIN-39's ability to activate terminal features of alternative neuronal identities. Loss of unc-3 causes a switch in the transcriptional targets of LIN-39, thereby alternative, not cholinergic MN-specific, terminal features become activated and locomotion defects occur. The strategy of a terminal selector preventing a transcriptional switch may constitute a general principle for safeguarding neuronal identity throughout life.

Anatomy and Physiology of Neurons in Layer 9 of the Chicken Optic Tectum.

Visual information in birds is to great extent processed in the optic tectum (TeO), a prominent laminated midbrain structure. Retinal input enters the TeO in its superficial layers, while output is limited to intermediate and deeper layers. In addition to visual information, the TeO receives multimodal input from the auditory and somatosensory pathway. The TeO gives rise to a major ascending tectofugal projection where neurons of tectal layer 13 project to the thalamic nucleus rotundus, which then projects to the entopallium. A second tectofugal projection system, called the accessory pathway, has however not been studied as thoroughly. Again, cells of tectal layer 13 form an ascending projection that targets a nucleus known as either the caudal part of the nucleus dorsolateralis posterior of the thalamus (DLPc) or nucleus uveaformis (Uva). This nucleus is known for multimodal integration and receives additional input from the lateral pontine nucleus (PL), which in turn receives projections from layer 8-15 of the TeO. Here, we studied a particular cell type afferent to the PL that consists of radially oriented neurons in layer 9. We characterized these neurons with respect to their anatomy, their retinal input, and the modulation of retinal input by local circuits. We found that comparable to other radial neurons in the tectum, cells of layer 9 have columnar dendritic fields and reach up to layer 2. Sholl analysis demonstrated that dendritic arborization concentrates on retinorecipient layers 2 and 4, with additional arborization in layers 9 and 10. All neurons recorded in layer 9 received retinal input via glutamatergic synapses. We analyzed the influence of modulatory circuits of the TeO by application of antagonists to γ-aminobutyric acid (GABA) and acetylcholine (ACh). Our data show that the neurons of layer 9 are integrated in a network under strong GABAergic inhibition, which is controlled by local cholinergic activation. Output to the PL and to the accessory tectofugal pathway thus appears to be under strict control of local tectal networks, the relevance of which for multimodal integration is discussed.

Restored presynaptic synaptophysin and cholinergic inputs contribute to the protective effects of physical running on spatial memory in aged mice.

The effects of prolonged physical training on memory performance and underlying presynaptic mechanisms were investigated in old C57BL/6 mice. Training via voluntary running wheels was initiated at 16 months of age and continued for 5 months (1 h per day, 5 days per week), followed by testing of learning and memory functions and counting of presynaptic puncta and cholinergic inputs in the hippocampus. Trained old mice were compared to their age-matched sedentary controls and adult controls. This training strategy improved hippocampal-dependent spatial memory function tested via a novel location task, and enhanced memory was accompanied by restored presynaptic puncta and cholinergic fibers in area CA1 and DG of the hippocampus in old mice. Particularly, the training selectively affected presynaptic vesicle protein synaptophysin but not growth associated protein GAP-43, and the increased number of synaptophysin puncta positively correlates with improved memory performance. To better understand the neurochemical mechanisms by which prolonged physical training protects against aging-related memory deficits, the cholinergic inputs to the hippocampus were compared among the three groups of mice and correlated with memory performance. While the running prevented age-related loss of cholinergic inputs, it has limited impact on the projection source cells in the medial septum-diagonal band (MS-DB). Importantly, cholinergic fibers in area CA1 and DG positively correlated with spatial memory function. These data suggest that the preservation of presynaptic inputs, particularly those involved in the integrity of memory performance, contributes critically to the beneficial effects of physical running initiated at an older age.

Three divisions of the mouse caudal striatum differ in the proportions of dopamine D1 and D2 receptor-expressing cells, distribution of dopaminergic axons, and composition of cholinergic and GABAergic interneurons.

The greater part of the striatum is composed of striosomes and matrix compartments, but we recently demonstrated the presence of a region that has a distinct structural organization in the ventral half of the mouse caudal striatum (Miyamoto et al. in Brain Struct Funct 223:4275-4291, 2018). This region, termed the tri-laminar part based upon its differential immunoreactivities for substance P and enkephalin, consists of medial, intermediate, and lateral divisions. In this study, we quantitatively analyzed the distributions of both projection neurons and interneurons in each division using immunohistochemistry. Two types of projection neurons expressing either the dopamine D1 receptor (D1R) or D2 receptor (D2R) showed complementary distributions throughout the tri-laminar part, but the proportions significantly differed among the three divisions. The proportion of D1R-expressing neurons in the medial, intermediate, and lateral divisions was 88.6 ± 8.2% (651 cells from 3 mice), 14.7 ± 3.8% (1025 cells), and 49.3 ± 4.5% (873 cells), respectively. The intermediate division was further characterized by poor innervation of tyrosine hydroxylase immunoreactive axons. The numerical density of choline acetyltransferase immunoreactive neurons differed among the three divisions following the order from the medial to lateral divisions. In contrast, PV-positive somata were distributed throughout all three divisions at a constant density. Two types of GABAergic interneurons labeled for nitric oxide synthase and calretinin showed the highest cell density in the medial division. The present results characterize the three divisions of the mouse caudal striatum as distinct structures, which will facilitate studies of novel functional loops in the basal ganglia.

Silica nanoparticle-exposure during neuronal differentiation modulates dopaminergic and cholinergic phenotypes in SH-SY5Y cells.

BACKGROUND: Silica-ε-polycaprolactone-nanoparticles (SiPCL-NPs) represent a promising tool for laser-tissue soldering in the brain. After release of the SiPCL-NPs in the brain, neuronal differentiation might be modulated. The present study was performed to determine effects of SiPCL-NP-exposure at different stages of neuronal differentiation in neuron-like SH-SY5Y cells. The resulting phenotypes were analyzed quantitatively and signaling pathways involved in neuronal differentiation and degeneration were studied. SH-SY5Y cells were differentiated with all-trans retinoic acid or staurosporine to obtain predominantly cholinergic or dopaminergic neurons. The resulting phenotype was analyzed at the end of differentiation with and without the SiPCL-NPs given at various times during differentiation. RESULTS: Exposure to SiPCL-NPs before and during differentiation led to a decreased cell viability of SH-SY5Y cells depending on the differentiation protocol used. SiPCL-NPs co-localized with the neuronal marker ß-3-tubulin but did not alter the morphology of these cells. A significant decrease in the number of tyrosine hydroxylase (TH) immunoreactive neurons was found in staurosporine-differentiated cells when SiPCL-NPs were added at the end of the differentiation. TH-protein expression was also significantly downregulated when SiPCL-NPs were applied in the middle of differentiation. Protein expression of the marker for the dopamine active transporter (DAT) was not affected by SiPCL-NPs. SiPCL-NP-exposure predominantly decreased the expression of the high-affinity choline transporter 1 (CHT1) when the NPs were given before the differentiation. Pathways involved in neuronal differentiation, namely Akt, MAP-K, MAP-2 and the neurodegeneration-related markers ß-catenin and GSK-3ß were not altered by NP-exposure. CONCLUSIONS: The decrease in the number of dopaminergic and cholinergic cells may implicate neuronal dysfunction, but the data do not provide evidence that pathways relevant for differentiation and related to neurodegeneration are impaired.

Differentiation of human glioblastoma U87 cells into cholinergic neuron.

To facilitate research methodologies for investigating the role of cholinergic nerves in many diseases, establishing an in vitro cholinergic neuron model is necessary. In this study, we investigated whether human glioblastoma U87 cells could be differentiated into cholinergic neurons in vitro. Sodium butyrate was used as the differentiation agent. The differentiated cells established by inducing U87 cells with sodium butyrate were named D-U87 cells. Immunofluorescence was used to label the neuronal markers MAP2, NF-M, and ChAT and the glial marker GFAP in D-U87 cells. Flow cytometry was used to measure cell cycle distribution in D-U87 cells. PCR, protein chip, and western blot assays were used to measure the expression levels of muscarinic cholinergic receptor 1 (M1), M4, ChAT, SYP and Akt. ELISA was used to measure neurotransmitter levels. As a result, we found that sodium butyrate induced U87 cell differentiation into cells with neuronal characteristics and increased not only the expression levels of the cholinergic neuron-related proteins M1, M4, ChAT and SYP in D-U87 cells but also the acetylcholine neurotransmitters in D-U87 cells. Moreover, the Akt protein expression in D-U87 cells was increased compared with that in U87 cells. Finally, we found that M1, M4, ChAT and SYP protein expression and acetylcholine secretion levels were significantly decreased in D-U87 cells after treatment with the Akt inhibitor MK-2206. These results demonstrate that D-U87 cells exhibit cholinergic neuron characteristics and that sodium butyrate induced U87 cell differentiation into cholinergic neuron partially through Akt signaling.

A whole-brain atlas of monosynaptic input targeting four different cell types in the medial prefrontal cortex of the mouse.

The local and long-range connectivity of cortical neurons are considered instrumental to the functional repertoire of the cortical region in which they reside. In cortical networks, distinct cell types build local circuit structures enabling computational operations. Computations in the medial prefrontal cortex (mPFC) are thought to be central to cognitive operation, including decision-making and memory. We used a retrograde trans-synaptic rabies virus system to generate brain-wide maps of the input to excitatory neurons as well as three inhibitory interneuron subtypes in the mPFC. On the global scale the input patterns were found to be mainly cell type independent, with quantitative differences in key brain regions, including the basal forebrain. Mapping of the local mPFC network revealed high connectivity between the different subtypes of interneurons. The connectivity mapping gives insight into the information that the mPFC processes and the structural architecture underlying the mPFC's unique functions.