Chronic inflammation with microglia senescence at basal forebrain: impact on cholinergic deficit in Alzheimer's brain haemodynamics.

Cholinergic innervation in the brain is involved in modulating neurovascular function including cerebral blood flow haemodynamics in response to neuronal activity. Cholinergic deficit is associated with pathophysiology in Alzheimer's disease, albeit the aetiology remains to be clarified. In the current study, neocortex cerebral blood flow response to acetylcholine was evaluated by Laser-Doppler Flowmetry (LDF) in 3xTgAD Alzheimer's disease model) and wild-type mice of two age groups. The peak of cerebral blood flow to acetylcholine (i.v.) from baseline levels (% ΔrCBF) was higher in young 3xTgAD versus in wild-type mice (48.35; 95% CI:27.03-69.67 versus 22.70; CI:15.5-29.91, P < 0.05); this was reversed in old 3xTgAD mice (21.44; CI:2.52-40.35 versus 23.25; CI:23.25-39). Choline acetyltransferase protein was reduced in neocortex, while cerebrovascular reactivity to acetylcholine was preserved in young 3×TgAD mice. This suggests endogenous acetylcholine deficit and possible cholinergic denervation from selected cholinergic nuclei within the basal forebrain. The early deposition of tauopathy moieties (mutant hTau and pTau181) and its coincidence in cholinergic cell clusters (occasionaly), were observed at the basal forebrain of 3xTgAD mice including substantia innominate, nucleus Basalis of Meynert and nucleus of horizontal limb diagonal band of Broca. A prominent feature was microglia interacting tauopathy and demonstrated a variety of morphology changes particularly when located in proximity to tauopathy. The microglia ramified phenotype was reduced as evaluated by the ramification index and Fractal analysis. Increased microglia senescence, identified as SASP (senescence-associated secretory phenotype), was colocalization with p16Ink4ɑ, a marker of irreversible cell-cycle arrest in old 3xTgAD versus wild-type mice (P = 0.001). The p16Ink4ɑ was also observed in neuronal cells bearing tauopathy within the basal forebrain of 3xTgAD mice. TNF-ɑ, the pro-inflammatory cytokine elevated persistently in microglia (Pearson's correlation coefficient = 0.62) and the loss of cholinergic cells in vulnerable basal forebrain environment, was indicated by image analysis in 3xTgAD mice, which linked to the cholinergic deficits in neocortex rCBF haemodynamics. Our study revealed the early change of CBF haemodynamics to acetylcholine in 3xTgAD model. As a major effector of brain innate immune activation, microglia SASP with age-related disease progression is indicative of immune cell senescence, which contributes to chronic inflammation and cholinergic deficits at the basal forebrain. Targeting neuroinflammation and senescence may mitigate cholinergic pathophysiology in Alzheimer's disease.

Septal stimulation attenuates hippocampal seizure with subregion specificity.

OBJECTIVE: Deep brain stimulation (DBS) is a promising approach for the treatment of epilepsy. However, the optimal target for DBS and underlying mechanisms are still not clear. Here, we compared the therapeutic effects of DBS on distinct septal subregions, aimed to find the precise targets of septal DBS and related mechanisms for the clinical treatment. METHODS: Assisted by behavioral test, electroencephalography (EEG) recording and analyzing, selectively neuronal manipulation and immunohistochemistry, we assessed the effects of DBS on the three septal subregions in kainic acid (KA)-induced mouse seizure model. RESULTS: DBS in the medial septum (MS) not only delayed generalized seizure (GS) development, but reduced the severity; DBS in the vertical diagonal band of Broca (VDB) only reduced the severity of GS, while DBS in the horizontal diagonal band of Broca (HDB) subregion showed no anti-seizure effect. Notably, DBS in the MS much more efficiently decreased abnormal activation of hippocampal neurons. EEG spectrum analysis indicated that DBS in the MS and VDB subregions mainly increased the basal hippocampal low-frequency (delta and theta) rhythm. Furthermore, ablation of cholinergic neurons in the MS and VDB subregions blocked the anti-seizure and EEG-modulating effects of septal DBS, suggesting the seizure-alleviating effect of DBS was dependent on local cholinergic neurons. SIGNIFICANCE: DBS in the MS and VDB, rather than HDB, attenuates hippocampal seizure by activation of cholinergic neurons-augmented hippocampal delta/theta rhythm. This may be of great therapeutic significance for the clinical treatment of epilepsy with septal DBS. PLAIN LANGUAGE SUMMARY: The optical target of deep brain stimulation in the septum is still not clear. This study demonstrated that stimulation in the medial septum and vertical diagonal band of Broca subregions, but not the horizontal diagonal band of Broca, could alleviate hippocampal seizure through cholinergic neurons-augmented hippocampal delta/theta rhythm. This study may shed light on the importance of precise regulation of deep brain stimulation therapy in treating epileptic seizures.

Distribution of cionin, a cholecystokinin/gastrin family peptide, and its receptor in the central nervous system of Ciona intestinalis type A.

The cholecystokinin (CCK)/gastrin family peptides are involved in regulation of feeding and digestion in vertebrates. In the ascidian Ciona intestinalis type A (Ciona robusta), cionin, a CCK/gastrin family peptide, has been identified. Cionin is expressed exclusively in the central nervous system (CNS). In contrast, cionin receptor expression has been detected in the CNS, digestive tract, and ovary. Although cionin has been reported to be involved in ovulation, its physiological function in the CNS remains to be investigated. To elucidate its neural function, in the present study, we analyzed the expression of cionin and cionin receptors in the CNS. Cionin was expressed mainly in neurons residing in the anterior region of the cerebral ganglion. In contrast, the gene expressin of the cionin receptor gene CioR1, was detected in the middle part of the cerebral ganglion and showed a similar expression pattern to that of VACHT, a cholinergic neuron marker gene. Moreover, CioR1 was found to be expressed in cholinergic neurons. Consequently, these results suggest that cionin interacts with cholinergic neurons as a neurotransmitter or neuromodulator via CioR1. This study provides insights into a biological role of a CCK/gastrin family peptide in the CNS of ascidians.

Analogs of α-conotoxin PnIC selectively inhibit α7ß2- over α7-only subtype nicotinic acetylcholine receptors via a novel allosteric mechanism.

This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and ß2 subunits (α7ß2-nAChR subtype). Basal forebrain cholinergic neurons express α7ß2-nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid-ß associated with early Alzheimer's disease. Additional work indicates that α7ß2-nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7ß2-nAChR from the closely related and more widespread homomeric α7-only-nAChR subtype. Functional screening using two-electrode voltage-clamp electrophysiology identified a family of α7ß2-nAChR-selective analogs of α-conotoxin PnIC (α-CtxPnIC). A combined electrophysiology, functional kinetics, site-directed mutagenesis, and molecular dynamics approach was used to further characterize the α7ß2-nAChR selectivity and site of action of these α-CtxPnIC analogs. We determined that α7ß2-nAChR selectivity of α-CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist-binding site shared between α7ß2- and α7-only-nAChR. As numerous previously identified α-Ctx ligands are competitive antagonists of orthosteric agonist-binding sites, this study profoundly expands the scope of use of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α-CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7ß2-nAChR and detailed investigations of their physiological roles.

Neuronal-type-specific epigenome editing to decrease SNCA expression: Implications for precision medicine in synucleinopathies.

Overexpression of SNCA has been implicated in the pathogenesis of synucleinopathies, particularly Parkinson's disease (PD) and dementia with Lewy bodies (DLB). While PD and DLB share some clinical and pathological similarities, each disease presents distinct characteristics, including the primary affected brain region and neuronal type. We aimed to develop neuronal-type-specific SNCA-targeted epigenome therapies for synucleinopathies. The system is based on an all-in-one lentiviral vector comprised of CRISPR-dSaCas9 and guide RNA (gRNA) targeted at SNCA intron 1 fused with a synthetic repressor molecule of Krüppel-associated box (KRAB)/ methyl CpG binding protein 2 (MeCp2) transcription repression domain (TRD). To achieve neuronal-type specificity for dopaminergic and cholinergic neurons, the system was driven by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) promoters, respectively. Delivering the system into human induced pluripotent stem cell (hiPSC)-derived dopaminergic and cholinergic neurons from a patient with the SNCA triplication resulted in efficient and neuronal-type-specific downregulation of SNCA-mRNA and protein. Furthermore, the reduction in SNCA levels by the gRNA-dSaCas9-repressor system rescued disease-related cellular phenotypes including Ser129-phophorylated α-synuclein, neuronal viability, and mitochondrial dysfunction. We established a novel neuronal-type-specific SNCA-targeted epigenome therapy and provided in vitro proof of concept using human-based disease models. Our results support the therapeutic potential of our system for PD and DLB and provide the foundation for further preclinical studies in animal models toward investigational new drug (IND) enablement and clinical trials.

Activation of medial septum cholinergic neurons restores cognitive function in temporal lobe epilepsy.

Cognitive impairment is the most common complication in patients with temporal lobe epilepsy with hippocampal sclerosis. There is no effective treatment for cognitive impairment. Medial septum cholinergic neurons have been reported to be a potential target for controlling epileptic seizures in temporal lobe epilepsy. However, their role in the cognitive impairment of temporal lobe epilepsy remains unclear. In this study, we found that patients with temporal lobe epilepsy with hippocampal sclerosis had a low memory quotient and severe impairment in verbal memory, but had no impairment in nonverbal memory. The cognitive impairment was slightly correlated with reduced medial septum volume and medial septum-hippocampus tracts measured by diffusion tensor imaging. In a mouse model of chronic temporal lobe epilepsy induced by kainic acid, the number of medial septum cholinergic neurons was reduced and acetylcholine release was reduced in the hippocampus. Furthermore, selective apoptosis of medial septum cholinergic neurons mimicked the cognitive deficits in epileptic mice, and activation of medial septum cholinergic neurons enhanced hippocampal acetylcholine release and restored cognitive function in both kainic acid- and kindling-induced epilepsy models. These results suggest that activation of medial septum cholinergic neurons reduces cognitive deficits in temporal lobe epilepsy by increasing acetylcholine release via projections to the hippocampus.

Scopolamine causes delirium-like brain network dysfunction and reversible cognitive impairment without neuronal loss.

Delirium is a severe acute neuropsychiatric syndrome that commonly occurs in the elderly and is considered an independent risk factor for later dementia. However, given its inherent complexity, few animal models of delirium have been established and the mechanism underlying the onset of delirium remains elusive. Here, we conducted a comparison of three mouse models of delirium induced by clinically relevant risk factors, including anesthesia with surgery (AS), systemic inflammation, and neurotransmission modulation. We found that both bacterial lipopolysaccharide (LPS) and cholinergic receptor antagonist scopolamine (Scop) induction reduced neuronal activities in the delirium-related brain network, with the latter presenting a similar pattern of reduction as found in delirium patients. Consistently, Scop injection resulted in reversible cognitive impairment with hyperactive behavior. No loss of cholinergic neurons was found with treatment, but hippocampal synaptic functions were affected. These findings provide further clues regarding the mechanism underlying delirium onset and demonstrate the successful application of the Scop injection model in mimicking delirium-like phenotypes in mice.

Whole-brain neural connectivity to cholinergic neurons in the nucleus basalis of Meynert.

The cholinergic neurons in the nucleus basalis of Meynert (NBM) are a key structure in cognition, the dysfunction of which is associated with various neurological disorders, especially dementias. However, the whole-brain neural connectivity to cholinergic neurons in the NBM remains to be further and comprehensively researched. Using virus-based, specific, retrograde, and anterograde tracing, we illustrated the monosynaptic inputs and axon projections of NBM cholinergic neurons in choline acetyltransferase (ChAT)-Cre transgenic mice. Our results showed that NBM cholinergic neurons received mainly inputs from the caudate putamen and the posterior limb of the anterior commissure in the subcortex. Moreover, the majority of cholinergic terminals from the NBM were observed in the cortex mantle, including the motor cortex, sensory cortex, and visual cortex. Interestingly, although NBM cholinergic neurons received input projections from the caudate putamen, interstitial nucleus of the posterior limb of the anterior commissure, and central amygdaloid nucleus, NBM cholinergic neurons sparsely sent axon projection to innervate these areas. Furthermore, primary motor cortex, secondary motor cortex, and primary somatosensory cortex received abundant inputs from the NBM but sent few outputs to the NBM. Taken together, our results reveal the detailed and specific connectivity of cholinergic neurons of the NBM and provide a neuroanatomic foundation for further studies to explore the important physiological functions of NBM cholinergic neurons.

Activation of cholinergic basal forebrain neurons improved cognitive functions in adult-onset hypothyroid mice.

Cognitive dysfunction is common in hypothyroid patients, even after undergoing sufficient levothyroxine (LT4) replacement therapy for euthyroid. Our previous studies indicated that cholinergic neurons might contribute to the decline of cognition in adult-onset hypothyroidism. Nevertheless, the role of the cellular and neural control of basal forebrain (BF) cholinergic neurons in hypothyroidism-induced cognitive impairments is unknown. Using transgenic mice that specifically expressed chemogenetic activators in their BF cholinergic neurons, we systematically investigated the role of BF cholinergic neurons in hypothyroidism-induced cognitive dysfunction by the combined approaches of patch clamp electrophysiology, behavioral testing, and immunohistochemistry. The results showed that LT4 treatment in the adult-onset hypothyroid mice reversed only 78 % of the BF cholinergic neurons to their normal values of electrophysiological properties. LT4 monotherapy did not rehabilitate cognitive function in the hypothyroid mice. Chemogenetic selective activation of the BF cholinergic neurons combined with LT4 treatment significantly improved learning and memory functions in the hypothyroid mice. In addition, chemogenetic activation of the cholinergic neurons induced the robust expression of c-Fos protein in the BF, prefrontal cortex (PFC), and hippocampus. This indicated that the BF cholinergic neurons improved learning and memory functions in the hypothyroid mice via the BF-PFC and BF-hippocampus pathways. In the hypothyroid C57BL/6 J mice, combined treatment via LT4 and donepezil, a cholinesterase inhibitor, significantly increased cognitive functions. The results suggested that the BF cholinergic neurons are critical for regulating learning and memory and reveal a novel pathophysiological mechanism for hypothyroidism-induced cognitive impairments.

Cholinergic REST-G9a gene repression through HMGB1-TLR4 neuroimmune signaling regulates basal forebrain cholinergic neuron phenotype.

Lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1) are Toll-like receptor (TLR4) agonists that activate proinflammatory neuroimmune signaling linked to loss of basal forebrain cholinergic neurons (BFCNs) and cognitive deficits. Loss of choline acetyltransferase immunoreactive (ChAT + IR) BFCNs is generally interpreted as cell death, but recent in vivo studies find anti-inflammatory interventions restore adolescent ethanol exposure-induced persistent loss of adult ChAT + IR neurons and cognitive deficits, suggesting proinflammatory signaling-induced reversible gene repression of ChAT in BFCNs. Using an ex vivo Wistar rat basal forebrain slice culture (FSC) model to investigate TLR4 involvement in repression of the BFCN phenotype, we report that direct TLR4 activation with LPS decreases expression of multiple BFCN markers in the absence of observable neuronal loss or cell death. Inhibition of HMGB1 blunts while inhibition of TLR4 blocks the LPS-induced loss of ChAT + IR neurons. TLR4 activation induces the transcriptional repressor RE1-silencing transcription factor (REST) and the methyltransferase G9a while increasing repressive histone 3 lysine 9 dimethylation and REST occupancy at cholinergic gene promoters. G9a inhibitors both prevent and reverse the LPS-induced loss of ChAT + IR whereas siRNA inhibition of REST blocks the LPS-induced loss of ChAT + IR BFCNs. These data suggest in vivo HMGB1-TLR4 signaling in BFCNs leads to a reversible loss of the cholinergic neuron phenotype through epigenetic gene repressive mechanisms.

The Response Dynamics and Function of Cholinergic and GABAergic Neurons in the Basal Forebrain During Olfactory Learning.

Modulation of neural circuits is essential for flexible sensory perception and decision-making in a changing environment. Cholinergic and GABAergic projections to the olfactory system from the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain are crucial for odor detection and olfactory learning. Although studies have demonstrated that HDB neurons respond during olfactory learning, how cholinergic and GABAergic neurons differ in their response dynamics and roles in olfactory learning remains unclear. In this study, we examined the response profiles of these two subpopulations of neurons during passive odor exposure and associative olfactory learning. We show that the excitatory responses in both cholinergic and GABAergic neurons tended to habituate during repeated passive odor exposure. However, while these habituated responses were also observed in GABAergic neurons during a go-go task, there was no such habituation in cholinergic neurons. Moreover, the responses to S+ and S- trials diverged in cholinergic neurons once mice learned a go/no-go task. Furthermore, the chemogenetic inactivation of cholinergic neurons in the HDB impaired odor discrimination. Together, these findings suggest that cholinergic neurons in the HDB reflect attention to positive reinforcement and may regulate odor discrimination via top-down inputs to the olfactory system.

Study of lead-induced neurotoxicity in cholinergic cells differentiated from bone marrow-derived mesenchymal stem cells.

The developing brain is susceptible to the neurotoxic effects of lead. Exposure to lead has main effects on the cholinergic system and causes reduction of cholinergic neuron function during brain development. Disruption of the cholinergic system by chemicals, which play important roles during brain development, causes of neurodevelopmental toxicity. Differentiation of stem cells to neural cells is recently considered a promising tool for neurodevelopmental toxicity studies. This study evaluated the toxicity of lead acetate exposure during the differentiation of bone marrow-derived mesenchyme stem cells (bone marrow stem cells, BMSCs) to CCholinergic neurons. Following institutional animal care review board approval, BMSCs were obtained from adult rats. The differentiating protocol included two stages that were pre-induction with ß-mercaptoethanol (BME) for 24 h and differentiation to cholinergic neurons with nerve growth factor (NGF) over 5 days. The cells were exposed to different lead acetate concentrations (0.1-100 µm) during three stages, including undifferentiated, pre-induction, and neuronal differentiation stages; cell viability was measured by MTT assay. Lead exposure (0.01-100 µg/ml) had no cytotoxic effect on BMSCs but could significantly reduce cell viability at 50 and 100 µm concentrations during pre-induction and neuronal differentiation stages. MAP2 and choline acetyltransferase (ChAT) protein expression were investigated by immunocytochemistry. Although cells treated with 100 µm lead concentration expressed MAP2 protein in the differentiation stages, they had no neuronal cell morphology. The ChAT expression was negative in cells treated with lead. The present study showed that differentiated neuronal BMSCs are sensitive to lead toxicity during differentiation, and it is suggested that these cells be used to study neurodevelopmental toxicity.

Conditional deletion of TMEM16A in cholinergic neurons of the medial habenula induces anhedonic-like behavior in mice.

The medial habenula (mHb), a subregion of the habenula, is involved in diverse brain functions, such as nicotine addiction, anxiety, and anhedonia. We recently reported that TMEM16A deficiency, a calcium-activated chloride channel, decreased the activity of mHb cholinergic neurons. Since downregulated activity in cholinergic neurons of the mHb is involved in anhedonia-like behavior, we here investigated whether conditional deletion of TMEM16A in mHb cholinergic neurons also displays anhedonia-like behavior. The conditional deletion of TMEM16A in the mHb cholinergic neurons of mice (TMEM16A cKO mice) was generated by crossing ChaT-Cre (+) with floxed TMEM16A f/f mice. TMEM16A cKO mice displayed significantly reduced social interaction, sucrose preference, female urine sniffing, and increased marble burying. These behavioral data suggest the potential role of TMEM16A in anhedonic-like behavior in mice. Taken together, the presented data suggest that TMEM16A-mediated mHb activity might be a therapeutic target for anhedonia-related symptoms.

Cis-regulatory elements of the cholinergic gene locus in the silkworm Bombyx mori.

Genes of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter are encoded in the same gene locus, called the cholinergic gene locus. They are essential in cholinergic neurons to maintain their functional phenotype. The genomic structure of the cholinergic gene locus is conserved among invertebrates to mammals. However, the cholinergic gene expression in a specific subset of neurons is unknown in insects except for Drosophila melanogaster. In this study, we analysed the upstream sequence of cholinergic gene locus in the silkworm Bombyx mori to identify specific cis-regulatory regions. We found multiple enhancer regions that are localized within 1 kb upstream of the cholinergic gene locus. The combination of promoter assays using small deletions and bioinformatic analysis among insect species illuminates two conserved sequences in the cis-regulatory region: TGACGTA and CCAAT, which are known as the cAMP response element and CAAT box, respectively. We found that dibutyryl-cAMP, an analogue of cAMP, influences the expression of ChAT in B. mori. Tissue-specific expression analysis of transcriptional factors identified potential candidates that control the cholinergic gene locus expression. Our investigation provides new insight into the regulation mechanism of cholinergic neuron-specific gene machinery in this lepidopteran insect.

Morphological characterization of cholinergic partition cells: A transmitter-specific tracing study by Cre/lox-dependent viral gene expression.

BACKGROUND: Partition cells are cholinergic interneurons located in lamina VII of the spinal cord. Some partition cells are the source of the cholinergic boutons, known as C-terminals or C-boutons, that modulate the activity of spinal motor neurons. Therefore, partition cells might play an important role in motor control. Previous studies categorized partition cells into three groups (medial, intermediate, and lateral partition cells) according to their distance from the central canal. However, the morphological characteristics of the three groups remain obscure. METHODS: To analyze the morphology of partition cells, we developed an efficient technique for visualization of specific neurons at single-cell level in particular positions using adenovirus vectors and Cre/lox mediated recombination. Cre/lox conditional vectors were injected into the spinal cord of choline acetyltransferase-Cre transgenic mice, and partition cells labeled by green fluorescent protein were reconstructed from histological serial sections at the single-cell level. RESULTS: This technique allowed for the visualization of partition cells at high resolution and revealed that partition cells had various patterns of dendrite orientations and fields. Most of the visualized partition cells had more than 60% of their dendrites located in lamina VII of the spinal cord. Partition cells had dendrites extending into various Rexed's laminae (V, VI, VII, VIII, IX, and X), but none of the cells had dendrites extending dorsal to lamina IV. The dendrites of partition cells terminated both ipsilaterally and bilaterally. We also found that C-terminals on motor neurons may be derived from the middle/outer group of partition cells. CONCLUSIONS: Our results indicated that partition cells have various morphological features of the dendritic pattern and may receive differential inputs. Our results suggested that C-terminals originate not only from medial but also from intermediate/lateral cholinergic partition cells. The present study suggests that intermediate/lateral partition cells modulate activities of motor neurons through C-terminal synapses.

P2X2 receptors supply extracellular choline as a substrate for acetylcholine synthesis.

Acetylcholine (ACh), an excitatory neurotransmitter, is biosynthesized from choline in cholinergic neurons. Import from the extracellular space to the intracellular environment through the high-affinity choline transporter is currently regarded to be the only source of choline for ACh synthesis. We recently demonstrated that the P2X2 receptor, through which large cations permeate, functions as an alternative pathway for choline transport in the mouse retina. In the present study, we investigated whether choline entering cells through P2X2 receptors is used for ACh synthesis using a recombinant system. When P2X2 receptors expressed on HEK293 cell lines were stimulated with ATP, intracellular ACh concentrations increased. These results suggest that P2X2 receptors function in a novel pathway that supplies choline for ACh synthesis.

Nerve Growth Factor Compromise in Down Syndrome.

The basal forebrain cholinergic system relies on trophic support by nerve growth factor (NGF) to maintain its phenotype and function. In Alzheimer's disease (AD), basal forebrain cholinergic neurons (BFCNs) undergo progressive atrophy, suggesting a deficit in NGF trophic support. Within the central nervous system, NGF maturation and degradation are tightly regulated by an activity-dependent metabolic cascade. Here, we present a brief overview of the characteristics of Alzheimer's pathology in Down syndrome (DS) with an emphasis on this NGF metabolic pathway's disruption during the evolving Alzheimer's pathology. Such NGF dysmetabolism is well-established in Alzheimer's brains with advanced pathology and has been observed in mild cognitive impairment (MCI) and non-demented individuals with elevated brain amyloid levels. As individuals with DS inexorably develop AD, we then review findings that support the existence of a similar NGF dysmetabolism in DS coinciding with atrophy of the basal forebrain cholinergic system. Lastly, we discuss the potential of NGF-related biomarkers as indicators of an evolving Alzheimer's pathology in DS.

Behavioural Fever Promotes an Inflammatory Reflex Circuit in Ectotherms.

BACKGROUND: The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms' thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified. METHODS: We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes' functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues. RESULTS: We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors' activity and key anti-inflammatory factors such as cytokine Il10 and Tgfß in target tissues. CONCLUSION: These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural-immune adaptation to infection and its protective relevance in ectotherm organisms.

Medial septum tau accumulation induces spatial memory deficit via disrupting medial septum-hippocampus cholinergic pathway.

Tau accumulation and cholinergic impairment are characteristic pathologies in Alzheimer's disease (AD). However, the causal role of tau accumulation in cholinergic lesion is elusive. Here, we observed an aberrant tau accumulation in the medial septum (MS) of 3xTg and 5xFAD mice, especially in their cholinergic neurons. Overexpressing hTau in mouse MS (MShTau ) for 6 months but not 3 months induced spatial memory impairment without changing object recognition and anxiety-like behavior, indicating a specific and time-dependent effect of MS-hTau accumulation on spatial cognitive functions. With increasing hTau accumulation, the MShTau mice showed a time-dependent cholinergic neuron loss with reduced cholinergic projections to the hippocampus. Intraperitoneal administration of donepezil, a cholinesterase inhibitor, for 1 month ameliorated the MS-hTau-induced spatial memory deficits with preservation of MS-hippocampal cholinergic pathway and removal of tau load; and the beneficial effects of donepezil was more prominent at low dose. Proteomics revealed that MS-hTau accumulation deregulated multiple signaling pathways with numerous differentially expressed proteins (DEPs). Among them, the vacuolar protein sorting-associated protein 37D (VP37D), an autophagy-related protein, was significantly reduced in MShTau mice; the reduction of VP37D was restored by donepezil, and the effect was more significant at low dose than high dose. These novel evidences reveal a causal role of tau accumulation in linking MS cholinergic lesion to hippocampus-dependent spatial cognitive damages as seen in the AD patients, and the new tau-removal and autophagy-promoting effects of donepezil may extend its application beyond simple symptom amelioration to potential disease modification.

Fluoxetine and Ketamine Reverse the Depressive but Not Anxiety Behavior Induced by Lesion of Cholinergic Neurons in the Horizontal Limb of the Diagonal Band of Broca in Male Rat.

The basal forebrain cholinergic system is involved in cognitive processes, but the role of the basal forebrain cholinergic system in depression is unknown. We investigated whether a lesion of cholinergic neurons in the horizontal limb of the diagonal band of Broca (HDB) produces depressive-like behavior and whether fluoxetine or ketamine inhibits such depressive-like behaviors. Here, in rats, we used 192 IgG-saporin to eliminate the cholinergic neurons of the HDB and evaluated depressive-like behaviors using a preference test for sucrose solution and the forced swimming test. Fourteen days after the injection of 192 IgG-saporin into the HDB, the rats exhibited a significantly fewer number of choline acetyltransferase positive cell density in HDB, accompanied with neuronal loss in the entire hippocampus. Meanwhile, these rats significantly reduced preference for sucrose solution, increased immobility time in the forced swimming test, reduced locomotor activity, decreased context dependent memory in fear conditioning and the time spent in the open arms of the plus-maze. A single dose of ketamine (10 mg/kg) increased the sucrose solution consumption, reduced the immobility time in the forced swim test (FST), and increased locomotor activity compared to vehicle-treated rats. Moreover, in rats that were continuously treated with fluoxetine (10 mg/kg/day for 11 days), the sucrose solution consumption increased, the immobility time in the FST decreased, and locomotor activity increased compared to vehicle-treated rats. The present results demonstrate that a lesion of HDB cholinergic neurons results in depressive-like and anxiety-like behaviors and that antidepressants such as fluoxetine or ketamine, can reverse these depressive-like behaviors but not anxiety-like behaviors, and suggest that a lesion of HDB cholinergic neurons and followed hippocampus damage may be involved in the pathogenesis of depression.