Impaired astrocytic synaptic function by peripheral cholesterol metabolite 27-hydroxycholesterol.

Astrocytes represent the most abundant cell type in the brain, where they play critical roles in synaptic transmission, cognition, and behavior. Recent discoveries show astrocytes are involved in synaptic dysfunction during Alzheimer's disease (AD). AD patients have imbalanced cholesterol metabolism, demonstrated by high levels of side-chain oxidized cholesterol known as 27-hydroxycholesterol (27-OH). Evidence from our laboratory has shown that elevated 27-OH can abolish synaptic connectivity during neuromaturation, but its effect on astrocyte function is currently unclear. Our results suggest that elevated 27-OH decreases the astrocyte function in vivo in Cyp27Tg, a mouse model of brain oxysterol imbalance. Here, we report a downregulation of glutamate transporters in the hippocampus of CYP27Tg mice together with increased GFAP. GLT-1 downregulation was also observed when WT mice were fed with high-cholesterol diets. To study the relationship between astrocytes and neurons, we have developed a 3D co-culture system that allows all the cell types from mice embryos to differentiate in vitro. We report that our 3D co-cultures reproduce the effects of 27-OH observed in 2D neurons and in vivo. Moreover, we found novel degenerative effects in astrocytes that do not appear in 2D cultures, together with the downregulation of glutamate transporters GLT-1 and GLAST. We propose that this transporter dysregulation leads to neuronal hyperexcitability and synaptic dysfunction based on the effects of 27-OH on astrocytes. Taken together, these results report a new mechanism linking oxysterol imbalance in the brain and synaptic dysfunction through effects on astrocyte function.

Different effects of CYP27A1 and CYP7B1 on cognitive function: Two mouse models in comparison.

The oxysterol 27-hydroxycholesterol (27OHC) is produced by the enzyme sterol 27-hydroxylase (Cyp27A1) and is mainly catabolized to 7α-Hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) by the enzyme cytochrome P-450 oxysterol 7α-hydroxylase (Cyp7B1). 27OHC is mostly produced in the liver and can reach the brain by crossing the blood-brain barrier. A large body of evidence shows that CYP27A1 overexpression and high levels of 27OHC have a detrimental effect on the brain, causing cognitive and synaptic dysfunction together with a decrease in glucose uptake in mice. In this work, we analyzed two mouse models with high levels of 27OHC: Cyp7B1 knock-out mice and CYP27A1 overexpressing mice. Despite the accumulation of 27OHC in both models, Cyp7B1 knock-out mice maintained intact learning and memory capacities, neuronal morphology, and brain glucose uptake over time. Neurons treated with the Cyp7B1 metabolite 7-HOCA did not show changes in synaptic genes and 27OHC-treated Cyp7B1 knock-out neurons could not counteract 27OHC detrimental effects. This suggests that 7-HOCA and Cyp7B1 deletion in neurons do not mediate the neuroprotective effects observed in Cyp7B1 knock-out animals. RNA-seq of neuronal nuclei sorted from Cyp7B1 knock-out brains revealed upregulation of genes likely to confer neuroprotection to these animals. Differently from Cyp7B1 knock-out mice, transcriptomic data from CYP27A1 overexpressing neurons showed significant downregulation of genes associated with synaptic function and several metabolic processes. Our results suggest that the differences observed in the two models may be mediated by the higher levels of Cyp7B1 substrates such as 25-hydroxycholesterol and 3ß-Adiol in the knock-out mice and that CYP27A1 overexpressing mice may be a more suitable model for studying 27-OHC-specific signaling. We believe that future studies on Cyp7B1 and Cyp27A1 will contribute to a better understanding of the pathogenic mechanisms of neurodegenerative diseases like Alzheimer's disease and may lead to potential new therapeutic approaches.

27-hydroxycholesterol promotes oligodendrocyte maturation: Implications for hypercholesterolemia-associated brain white matter changes.

Oxidized cholesterol metabolite 27-hydroxycholesterol (27-OH) is a potential link between hypercholesterolemia and neurodegenerative diseases since unlike peripheral cholesterol, 27-OH is transported across the blood-brain barrier. However, the effects of high 27-OH levels on oligodendrocyte function remain unexplored. We hypothesize that during hypercholesterolemia 27-OH may impact oligodendrocytes and myelin and thus contribute to the disconnection of neural networks in neurodegenerative diseases. To test this idea, we first investigated the effects of 27-OH in cultured oligodendrocytes and found that it induces cell death of immature O4+ /GalC+ oligodendrocytes along with stimulating differentiation of PDGFR+ oligodendrocyte progenitors (OPCs). Next, transgenic mice with increased systemic 27-OH levels (Cyp27Tg) underwent behavioral testing and their brains were immunohistochemically stained and lysed for immunoblotting. Chronic exposure to 27-OH in mice resulted in increased myelin basic protein (MBP) but not 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) or myelin oligodendrocyte glycoprotein (MOG) levels in the corpus callosum and cerebral cortex. Intriguingly, we also found impairment of spatial learning suggesting that subtle changes in myelinated axons of vulnerable areas like the hippocampus caused by 27-OH may contribute to impaired cognition. Finally, we found that 27-OH levels in cerebrospinal fluid from memory clinic patients were associated with levels of the myelination regulating CNPase, independently of Alzheimer's disease markers. Thus, 27-OH promotes OPC differentiation and is toxic to immature oligodendrocytes as well as it subtly alters myelin by targeting oligodendroglia. Taken together, these data indicate that hypercholesterolemia-derived higher 27-OH levels change the oligodendrocytic capacity for appropriate myelin remodeling which is a crucial factor in neurodegeneration and aging.

Evaluation of the therapeutic efficacy of dressings with ZnO nanoparticles in the treatment of diabetic foot ulcers.

Type 2 diabetes (T2D) in developed countries have a prevalence of 11% with diabetic foot infections as the most common cause of hospitalization and amputation. To achieve healing of the diabetic foot ulcer wounds, appropriate dressings are essential and their effectiveness can be enhanced with nanoparticles, nevertheless ideal combinations of dressing composition and nanodrugs require further testing in humans. We have developed a calcium alginate dressings with ZnO nanoparticles (CAZnODs) for the treatment of diabetic foot ulcers in human patients. To test the efficacy of CAZnODs we designed a randomized controlled clinical experiment on 26 T2D patients with foot ulcers. The patients were randomized into two groups: G1 treatment with calcium alginate with NPs (G1; n = 16), and group 2 received the treatment without NPs (G2, n = 10). The bandage change was performed every 48 h The duration of the protocol was established at 10 weeks. Here, we report healing was achieved in patients, with 75% wound closure in G1 under treatment with NPs of calcium alginate versus 71% in G2 (calcium alginate without NPs) (p = 0.011). The average healing time was 48 days in G1 and 72 days in G2. Our data shows that CAZnODs were well tolerated and did not interfere with the wound healing process. The final wound area and time of healing support the hypothesis that the use of calcium alginate dressings with nanoparticles may induce better tissue regeneration while avoiding T2D complications such as secondary infections.

Development of zinc oxide/hydroxyapatite/poly(D,L-lactic acid) fibrous scaffold for tissue engineering applications.

Scaffolds based on polymeric fibers represent an engaging biomedical device due to their particular morphology and similarity with extracellular matrices. The biggest challenge to use fibrous materials in the biomedical field is related to their favorable platform for the adhesion of pathogenic microorganisms. Therefore, their optimum performance not only depends on their bioactive potential but also on their antimicrobial properties. The aim of this work was the design of antimicrobial (zinc oxide, ZnO) and bioactive (hydroxyapatite, Hap) fibrous materials using poly(D, L-lactic acid) (PDLLA) as the polymer fiber substrate. Fiber based composite scaffolds were developed using the Forcespinning® technique. For analysis purposes, the morphological, thermal, antimicrobial and biological properties of the fibrous hybrid system obtained at a concentration of 5 wt% of ZnO and 5 wt% of Hap were studied. The incorporation of the aforementioned nanoparticles (NPs) mixture in PDLLA led to an increase in viscosity and a pseudo-plastic tendency of the precursor solution, which caused an increase in fiber diameters and their dispersion of values. Small cavities and certain roughness were the main surface morphology observed on the fibers before and after NPs incorporation. The fiber thermal stability decreased due to the presence of the NPs. The antimicrobial properties of the hybrid fibrous scaffold presented a growth inhibition (GI) of 70 and 85% for E. coli and S. aureus strains, respectively. Concerning the osteoblast-cell compatibility, PDLLA and hybrid PDLLA scaffold showed low toxicity (cell viabilities above 80%), allowing cell growth inside its three-dimension structure and favorable cell morphology extended along the fibers. This behavior suggests a promising potential of this hybrid PDLLA scaffold for bone application.

Defects of Nutrient Signaling and Autophagy in Neurodegeneration.

Neurons are post-mitotic cells that allocate huge amounts of energy to the synthesis of new organelles and molecules, neurotransmission and to the maintenance of redox homeostasis. In neurons, autophagy is not only crucial to ensure organelle renewal but it is also essential to balance nutritional needs through the mobilization of internal energy stores. A delicate crosstalk between the pathways that sense nutritional status of the cell and the autophagic processes to recycle organelles and macronutrients is fundamental to guarantee the proper functioning of the neuron in times of energy scarcity. This review provides a detailed overview of the pathways and processes involved in the balance of cellular energy mediated by autophagy, which when defective, precipitate the neurodegenerative cascade of Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or Alzheimer's disease.

Functionally-distinct pyramidal cell subpopulations during gamma oscillations in mouse hippocampal area CA3.

Gamma oscillations (γ-oscillations) in hippocampal area CA3 are essential for memory function. Particularly, CA3 is involved in the memory related process pattern completion, which is linked with the γ-oscillations in human hippocampus. Recent studies suggest that heterogeneity in the functional properties of pyramidal cells (PCs) in CA3 plays an important role in hippocampal function. By performing concomitant recordings of PC activity and network γ-oscillations in CA3 we found three functionally-different PC subpopulations. PCs with high spike-frequency adaptation (hAPC) have the strongest action potential gamma phase-coupling, PCs with low adaptation (lAPC) show lower phase-coupling and PCs displaying a burst-firing pattern (BPC) remained quiescent. In addition, we discovered that hAPC display the highest excitatory/inhibitory drive, followed by lAPC, and lastly BPC. In conclusion, our data advance the hypothesis that PCs in CA3 are organized into subpopulations with distinct functional roles for cognition-relevant network dynamics and provide new insights in the physiology of hippocampus.

Brain Renin-Angiotensin System as Novel and Potential Therapeutic Target for Alzheimer's Disease.

The activation of the brain renin-angiotensin system (RAS) plays a pivotal role in the pathophysiology of cognition. While the brain RAS has been studied before in the context of hypertension, little is known about its role and regulation in relation to neuronal function and its modulation. Adequate blood flow to the brain as well as proper clearing of metabolic byproducts become crucial in the presence of neurodegenerative disorders such as Alzheimer's disease (AD). RAS inhibition (RASi) drugs that can cross into the central nervous system have yielded unclear results in improving cognition in AD patients. Consequently, only one RASi therapy is under consideration in clinical trials to modify AD. Moreover, the role of non-genetic factors such as hypercholesterolemia in the pathophysiology of AD remains largely uncharacterized, even when evidence exists that it can lead to alteration of the RAS and cognition in animal models. Here we revise the evidence for the function of the brain RAS in cognition and AD pathogenesis and summarize the evidence that links it to hypercholesterolemia and other risk factors. We review existent medications for RASi therapy and show research on novel drugs, including small molecules and nanodelivery strategies that can target the brain RAS with potential high specificity. We hope that further research into the brain RAS function and modulation will lead to innovative therapies that can finally improve AD neurodegeneration.

Hypercholesterolemia and 27-Hydroxycholesterol Increase S100A8 and RAGE Expression in the Brain: a Link Between Cholesterol, Alarmins, and Neurodegeneration.

Alterations in cholesterol metabolism in the brain have a major role in the physiology of Alzheimer's disease (AD). Oxysterols are cholesterol metabolites with multiple implications in memory functions and in neurodegeneration. Previous studies have shown detrimental effects of cholesterol metabolites in neurons, but its effect in glial cells is unknown. We used a high-fat/high-cholesterol diet in mice to study the effects of hypercholesterolemia over the alarmin S100A8 cascade in the hippocampus. Using CYP27Tg, a transgenic mouse model, we show that the hypercholesterolemia influence on the brain is mediated by the excess of 27-hydroxycholesterol (27-OH), a cholesterol metabolite. We also employed an acute model of 27-OH intraventricular injection in the brain to study RAGE and S100A8 response. We used primary cultures of neurons and astrocytes to study the effect of high levels of 27-OH over the S100A8 alarmin cascade. We report that a high-fat/high-cholesterol diet leads to an increase in S100A8 production in the brain. In CYP27Tg, we report an increase of S100A8 and its receptor RAGE in the hippocampus under elevated 27-OH in the brain. Using siRNA, we found that 27-OH upregulation of RAGE in astrocytes and neurons is mediated by the nuclear receptor RXRγ. Silencing RXRγ in neurons prevented 27-OH-mediated upregulation of RAGE. These results show that S100A8 alarmin and RAGE respond to high levels of 27-OH in the brain in both neurons and astrocytes through RXRγ. Our study supports the notion that 27-OH mediates detrimental effects of hypercholesterolemia to the brain via alarmin signaling.

Impaired spike-gamma coupling of area CA3 fast-spiking interneurons as the earliest functional impairment in the AppNL-G-F mouse model of Alzheimer's disease.

In Alzheimer's disease (AD) the accumulation of amyloid-ß (Aß) correlates with degradation of cognition-relevant gamma oscillations. The gamma rhythm relies on proper neuronal spike-gamma coupling, specifically of fast-spiking interneurons (FSN). Here we tested the hypothesis that decrease in gamma power and FSN synchrony precede amyloid plaque deposition and cognitive impairment in AppNL-G-F knock-in mice (AppNL-G-F). The aim of the study was to evaluate the amyloidogenic pathology progression in the novel AppNL-G-F mouse model using in vitro electrophysiological network analysis. Using patch clamp of FSNs and pyramidal cells (PCs) with simultaneous gamma oscillation recordings, we compared the activity of the hippocampal network of wild-type mice (WT) and the AppNL-G-F mice at four disease stages (1, 2, 4, and 6 months of age). We found a severe degradation of gamma oscillation power that is independent of, and precedes Aß plaque formation, and the cognitive impairment reported previously in this animal model. The degradation correlates with increased Aß1-42 concentration in the brain. Analysis on the cellular level showed an impaired spike-gamma coupling of FSN from 2 months of age that correlates with the degradation of gamma oscillations. From 6 months of age PC firing becomes desynchronized also, correlating with reports in the literature of robust Aß plaque pathology and cognitive impairment in the AppNL-G-F mice. This study provides evidence that impaired FSN spike-gamma coupling is one of the earliest functional impairment caused by the amyloidogenic pathology progression likely is the main cause for the degradation of gamma oscillations and consequent cognitive impairment. Our data suggests that therapeutic approaches should be aimed at restoring normal FSN spike-gamma coupling and not just removal of Aß.

High levels of 27-hydroxycholesterol results in synaptic plasticity alterations in the hippocampus.

Alterations in brain cholesterol homeostasis in midlife are correlated with a higher risk of developing Alzheimer's disease (AD). However, global cholesterol-lowering therapies have yielded mixed results when it comes to slowing down or preventing cognitive decline in AD. We used the transgenic mouse model Cyp27Tg, with systemically high levels of 27-hydroxycholesterol (27-OH) to examine long-term potentiation (LTP) in the hippocampal CA1 region, combined with dendritic spine reconstruction of CA1 pyramidal neurons to detect morphological and functional synaptic alterations induced by 27-OH high levels. Our results show that elevated 27-OH levels lead to enhanced LTP in the Schaffer collateral-CA1 synapses. This increase is correlated with abnormally large dendritic spines in the stratum radiatum. Using immunohistochemistry for synaptopodin (actin-binding protein involved in the recruitment of the spine apparatus), we found a significantly higher density of synaptopodin-positive puncta in CA1 in Cyp27Tg mice. We hypothesize that high 27-OH levels alter synaptic potentiation and could lead to dysfunction of fine-tuned processing of information in hippocampal circuits resulting in cognitive impairment. We suggest that these alterations could be detrimental for synaptic function and cognition later in life, representing a potential mechanism by which hypercholesterolemia could lead to alterations in memory function in neurodegenerative diseases.

Thioredoxin-80 protects against amyloid-beta pathology through autophagic-lysosomal pathway regulation.

Aggregation and accumulation of amyloid beta (Aß) are believed to play a key role in the pathogenesis of Alzheimer's disease (AD). We previously reported that Thioredoxin-80 (Trx80), a truncated form of Thioredoxin-1, prevents the toxic effects of Aß and inhibits its aggregation in vitro. Trx80 levels were found to be dramatically reduced both in the human brain and cerebrospinal fluid of AD patients. In this study, we investigated the effect of Trx80 expression using in vivo and in vitro models of Aß pathology. We developed Drosophila melanogaster models overexpressing either human Trx80, human Aß42, or both Aß42/Trx80 in the central nervous system. We found that Trx80 expression prevents Aß42 accumulation in the brain and rescues the reduction in life span and locomotor impairments seen in Aß42 expressing flies. Also, we show that Trx80 induces autophagosome formation and reverses the inhibition of Atg4b-Atg8a/b autophagosome formation pathway caused by Aß42. These effects were also confirmed in human neuroblastoma cells. These results give insight into Trx80 function in vivo, suggesting its role in the autophagosome biogenesis and thus in Aß42 degradation. Our findings put Trx80 on the spotlight as an endogenous agent against Aß42-induced toxicity in the brain suggesting that strategies to enhance Trx80 levels in neurons could potentially be beneficial against AD pathology in humans.

Restoring synaptic function through multimodal therapeutics.

Alzheimer's disease (AD) is the major form of dementia and a growing epidemic for which no disease-modifying treatments exist. AD is characterized by the early loss of synapses in the brain and, at later stages, neuronal death accompanied with progressive loss of cognitive functions. Here we focus on the mechanisms involved in the maintenance of the synapse and how their perturbation leads to synaptic loss. We suggest treatment strategies that particularly target energy metabolism in terms of cholesterol and glucose biochemistry in neurons and astrocytes We also discuss the potential of restoring impaired protein homeostasis through autophagy. These pathways are analyzed from a basic science perspective and suggest new avenues for discovery. We also propose several targets for both basic and translational therapeutics in these pathways and provide perspective on future AD treatment.

Alterations in cholesterol metabolism as a risk factor for developing Alzheimer's disease: Potential novel targets for treatment.

Alzheimer's disease (AD) is the most common form of dementia and it is characterized by the deposition of amyloid-ß (Aß) plaques and neurofibrillary tangles in the brain. However, the complete pathogenesis of the disease is still unknown. High level of serum cholesterol has been found to positively correlate with an increased risk of dementia and some studies have reported a decreased prevalence of AD in patients taking cholesterol-lowering drugs. Years of research have shown a strong correlation between blood hypercholesterolemia and AD, however cholesterol is not able to cross the Blood Brain Barrier (BBB) into the brain. Cholesterol lowering therapies have shown mixed results in cognitive performance in AD patients, raising questions of whether brain cholesterol metabolism in the brain should be studied separately from peripheral cholesterol metabolism and what their relationship is. Unlike cholesterol, oxidized cholesterol metabolites known as oxysterols are able to cross the BBB from the circulation into the brain and vice-versa. The main oxysterols present in the circulation are 24S-hydroxycholesterol and 27-hydroxycholesterol. These oxysterols and their catalysing enzymes have been found to be altered in AD brains and there is evidence indicating their influence in the progression of the disease. This review gives a broad perspective on the relationship between hypercholesterolemia and AD, cholesterol lowering therapies for AD patients and the role of oxysterols in pathological and non-pathological conditions. Also, we propose cholesterol metabolites as valuable targets for prevention and alternative AD treatments.

27-Hydroxycholesterol Induces Aberrant Morphology and Synaptic Dysfunction in Hippocampal Neurons.

Hypercholesterolemia is a risk factor for neurodegenerative diseases, but how high blood cholesterol levels are linked to neurodegeneration is still unknown. Here, we show that an excess of the blood-brain barrier permeable cholesterol metabolite 27-hydroxycholesterol (27-OH) impairs neuronal morphology and reduces hippocampal spine density and the levels of the postsynaptic protein PSD95. Dendritic spines are the main postsynaptic elements of excitatory synapses and are crucial structures for memory and cognition. Furthermore, PSD95 has an essential function for synaptic maintenance and plasticity. PSD95 synthesis is controlled by the REST-miR124a-PTBP1 axis. Here, we report that high levels of 27-OH induce REST-miR124a-PTBP1 axis dysregulation in a possible RxRγ-dependent manner, suggesting that 27-OH reduces PSD95 levels through this mechanism. Our results reveal a possible molecular link between hypercholesterolemia and neurodegeneration. We discuss the possibility that reduction of 27-OH levels could be a useful strategy for preventing memory and cognitive decline in neurodegenerative disorders.

Ano1 is a better marker than c-Kit for transcript analysis of single interstitial cells of Cajal in culture.

The interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.

Expression of P2X3 and P2X 5 myenteric receptors varies during the intestinal postnatal development in the guinea pig.

P2X3 receptor expression in various tissues appears to be modulated by age. In the present study, we used single cell RT-PCR to determine the number of P2X3 positive myenteric neurons at different stages of guinea pig postnatal development, and we tested if similar changes also occur to other myenteric P2X receptors. Moreover, we carried out whole-cell recordings using Patch Clamp techniques to determine possible changes in P2X receptors sensitivity to ATP and α,ß-methylene ATP (α,ß-meATP) between newborn and adult animals. Our data indicate that P2X3 subunit transcripts are present in a larger number of myenteric neurons from newborn guinea pigs whereas P2X5 mRNA is found more frequently in adults. Expression of P2X2 and P2X4 transcripts does not change during postnatal development. In newborn animals, virtually all neurons expressing P2X3 also expressed P2X2 transcripts. This is important because these two subunits are known to form heteromeric channels. ATP potency to activate P2X receptors in neurons of both newborn and adult animals was the same. α,ß-meATP, a known P2X3 receptor agonist, induces only a marginal current despite the fact of the higher presence of P2X3 subunits in newborns. These findings imply that P2X3 subunits are mainly forming heteromeric, α,ß-meATP insensitive channels perhaps because P2X3 contributes with only one subunit to the heterotrimers while the other subunits could be P2X2, P2X4, or P2X5.

Cholinergic signalling-regulated KV7.5 currents are expressed in colonic ICC-IM but not ICC-MP.

Interstitial cells of Cajal (ICC) and the enteric nervous system orchestrate the various rhythmic motor patterns of the colon. Excitation of ICC may evoke stimulus-dependent pacemaker activity and will therefore have a profound effect on colonic motility. The objective of the present study was to evaluate the potential role of K(+) channels in the regulation of ICC excitability. We employed the cell-attached patch clamp technique to assess single channel activity from mouse colon ICC, immunohistochemistry to determine ICC K(+) channel expression and single cell RT-PCR to identify K(+) channel RNA. Single channel activity revealed voltage-sensitive K(+) channels, which were blocked by the KV7 blocker XE991 (n = 8), which also evoked inward maxi channel activity. Muscarinic acetylcholine receptor stimulation with carbachol inhibited K(+) channel activity (n = 8). The single channel conductance was 3.4 ± 0.1 pS (n = 8), but with high extracellular K(+), it was 18.1 ± 0.6 pS (n = 22). Single cell RT-PCR revealed Ano1-positive ICC that were positive for KV7.5. Double immunohistochemical staining of colons for c-Kit and KV7.5 in situ revealed that intramuscular ICC (ICC-IM), but not ICC associated with the myenteric plexus (ICC-MP), were positive for KV7.5. It also revealed dense cholinergic innervation of ICC-IM. ICC-IM and ICC-MP networks were found to be connected. We propose that the pacemaker network in the colon consists of both ICC-MP and ICC-IM and that one way of exciting this network is via cholinergic KV7.5 channel inhibition in ICC-IM.