Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and a-linolenic acid. EFAs are converted into x3-and x6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive. Here, we show that deletion of D6-fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2À/À mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA-substituted membrane phospholipids in Sertoli cell polarity and blood-testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2À/À mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.
In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.
Targeted deletion of the stearoyl-CoA desaturase 1 gene (scd1) in mouse causes obesity resistance and a severe skin phenotype. Here, we demonstrate that SCD1 deficiency disrupts the epidermal lipid barrier and leads to uncontrolled transepidermal water loss, breakdown of adaptive thermoregulation and cold resistance, as well as a metabolic wasting syndrome. The loss of omega-hydroxylated very long-chain fatty acids (VLCFA) and ceramides substituted with omega-hydroxylated VLCFA covalently linked to corneocyte surface proteins leads to the disruption of the epidermal lipid barrier in scd1-/- mutants. Artificial occlusion of the skin by topical lipid application largely reconstituted the epidermal barrier and also reversed dysregulation of thermogenesis and cold resistance, as well as the metabolic disturbances. Interestingly, SCD1 deficiency abolished expression of the key transcription factor Lef1, which is essential for interfollicular epidermis, sebaceous glands, and hair follicle development. Finally, the occurrence of SCD1 and a newly described hSCD5 (ACOD4) gene in humans suggests that the scd1-/- mouse mutant might be a valuable animal model for the study of human skin diseases associated with epidermal barrier defects.
Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, associated with retarded chondrocyte development and enchondral ossification in the epiphyseal growth plate.
Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, for example, wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M 4 and M1 mAChRs, respectively. At ~ 1 mM, ACh depolarized exclusively CT PCs via α4β2 subunit-containing nAChRs without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was suppressed via M4 mAChR activation but enhanced by nAChRs via α4β2 nAChRs when the presynaptic neuron was a CT PC. Thus, in L6A, the interaction of mAChRs and nAChRs results in an altered excitability and synaptic release, effectively strengthening CT output while weakening CC synaptic signaling.
22Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, e.g. 23 wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors 24 (mAChRs) and nicotinic AChRs (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types 25 of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in 26 corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M4 and M1 mAChRs, 27 respectively. At ~1 mM ACh depolarized exclusively CT PCs via α4β2 subunit-containing nAChRs 28 without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but 29 increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was 30 suppressed via M4 mAChR activation but enhanced by nAChRs via α4β2 nAChRs when the 31 presynaptic neuron was a CT PC. Thus, in layer 6A the interaction of mAChRs and nAChRs results 32 in an altered excitability and synaptic release, effectively strengthening corticothalamic output 33 while weakening corticocortical synaptic signaling. 34 35 Keywords: barrel cortex, layer 6, pyramidal cells, acetylcholine, muscarinic receptors, nicotinic 36 receptors, corticocortical, corticothalamic 37Acetylcholine (ACh) has been shown to play a major role in memory processing, arousal, attention 40 and sensory signaling 1-7 . It has been demonstrated that the ACh concentration in the cerebrospinal 41 fluid increases during wakefulness and sustained attention 8,9 . In the neocortex release of ACh occurs 42 predominately via afferents originating from cholinergic neurons in the nucleus basalis of Meynert 43 of the basal forebrain 10-12 ; their terminals are densely distributed throughout all neocortical layers 13-44 15 . A classical view is that ACh invariably increases the excitability of excitatory neurons in 45 neocortex 16-20. However, a persistent hyperpolarization in layer 4 (L4) excitatory neurons was 46 found in somatosensory cortex 21,22 . This layer-specific cholinergic modulation may contribute to 47 improving the cortical signal-to-noise ratio [23][24][25] . 48Although extensive studies have been conducted on the cholinergic modulation of neocortical 49 excitatory neurons, the action of ACh on the layer 6 (L6) microcircuitry has not been systematically 50 investigated. Two main pyramidal cell (PC) classes exist in cortical layer 6, namely corticothalamic 51 (CT) and corticocortical (CC) PCs. These two neuron types differ in their axonal projection patterns, 52 dendritic morphological features, electrophysiological properties and expression of molecular 53 markers 26-30 . CC PCs have no subcortical target and send intracortical projections mainly within the 54 infra-granular layers 28 ; CT PCs, in contrast, have few axons distributed in cortex and send 55 projections directly back to the thalamus thereby contributing to a feedback control of sensory 56 input 31-35 . The question how the function of these two classes of L6 PCs is modulated by ACh has ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.